Table Of Content

GE Healthcare A f fi n it y C h r o m a t o g r a p h y – V o l. 2 : T a g g e d P r o t e in s www.gelifesciences.com GE, GE monogram, Amersham, ÄKTA, Biacore, Capto, ECL, ECL Plex, ECL Select, ExcelGel, GraviTrap, GSTPrep, GSTrap, HiLoad, HiPrep, HiScale, HiScreen, HisPrep, HisTrap, HiTrap, Hybond, ImageQuant, MabSelect, MabSelect SuRe, MabSelect Xtra, MBPTrap, MidiTrap, MiniTrap, Mono Q, Multiphor, MultiTrap, PhastGel, PhastSystem, PreScission, PrimeView, Protran, Rainbow, RESOURCE, Sephacryl, Sephadex, Sepharose, SpinTrap, SOURCE, StrepTrap, Superdex, Superloop, Tricorn, and UNICORN are trademarks of General Electric Company. AcTEV, GIBCO, High Five, Novex, and SimplyBlue are trademarks of Life Technologies Corp. Amicon, Benzonase, BugBuster, Diaflo, and Milli-Q are trademarks of Merck KGaA. BCA and HisProbe are trademarks of Thermo Fisher Scientific. Coomassie is a trademark of Imperial Chemical Industries, Ltd. CYMAL and SAFC are trademarks of Affymetrix Inc. Eppendorf is a trademark of Eppendorf AG. EX-CELL and SAFC are trademarks of Sigma-Aldrich Co. Gel Doc is a trademark of BioRad Laboratories Inc. GenBank is a trademark of US Dept. of Health & Human Services. Kathon is a trademark of Dow Chemical Co. MODDE is a trademark of Umetrics AB. Pefabloc is a trademark of Pentapharm Ltd. Strep-Tactin and Strep-tag are trademarks of IBA GmbH. Superflow is a trademark of Sterogene Bioseparations Inc. TALON is a registered trademark of Clontech Laboratories Inc. Tween is a trademark of Uniqema Americas LLC. Vibracell is a trademark of Sonics & Materials, Inc. Vivaspin is a trademark of Sartorius Stedim Biotech GmbH. ECL Prime: This product or portions thereof is manufactured and sold under license from Cyanagen Srl and is subject Affinity Chromatography of US patent 7,855,287, US Patent 7,803,573, and US Patent 9,040,252, together with other equivalent granted patents and patent applications in other countries. IMAC Sepharose products, Ni Sepharose products and Fe Sepharose products: These products are sold under a license from Sigma-Aldrich under patent number EP 1276716 (Metal chelating compositions) and equivalent patents and Vol. 2: Tagged Proteins patent applications in other countries. StrepTrap HP and StrepTactin Sepharose High Performance: These products are covered by US patent number GE Healthcare Bio-Sciences AB 6,103,493 and equivalent patents and patent applications in other countries. The purchase of StrepTrap HP and StrepTactin Sepharose High Performance includes a license under such patents for nonprofit and in-house research Björkgatan 30 only. Please contact IBA ([email protected]) for further information on licenses for commercial use of StrepTactin. 751 84 Uppsala TALON Superflow and HiTrap TALON: TALON is a registered trademark of Clontech Laboratories Inc. © 2000–2016 General Electric Company. First published Dec 2000. Sweden All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information GE Healthcare UK Ltd, Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA, UK GE Healthcare Bio-Sciences Corp. 100 Results Way, Marlborough, MA 01752, USA GE Healthcare Dharmacon, Inc., 2650 Crescent Dr., Lafayette, CO 80026, USA imagination atH ywClonoe Larbkoratories, Inc., 925 W 1800 S, Logan, UT 84321, USA imagination at work GE Healthcare Europe GmbH, Munzinger Strasse 5, D-79111 Freiburg, Germany GE Healthcare Japan Corporation, Sanken Bldg. 3-25-1, Hyakunincho, Shinjuku-ku, Tokyo 169-0073, Japan For local office contact information, visit www.gelifesciences.com/contact imagination at work 18114275 AF 04/2016 Handbooks from GE Healthcare Life Sciences For more information refer to www.gelifesciences.com/handbooks GE Healthcare Affinity Chromatography GLiEfe H Secaieltnhcceasre High-throughput Process GLiEfe H Secaieltnhcceasre Protein Sample Vol. 1: Antibodies Development with Preparation 18103746 HPwriiogthch e-Ptsrhser DDouiecgvtoehrlpo™up Ptmlaetenst PreDictor Plates Handbook Principles and Methods Principles and Methods 28988741 28940358 Protein Sample AVofl.fi 1n: Ainttyib oCdhiesromatography PHarnedbpooakration GE Healthcare Affinity Chromatography GLiEfe H Secaieltnhcceasre Hydrophobic Interaction GLiEfe H Secaieltnhcceasre Purifying Challenging Vol. 2: Tagged Proteins and Reversed Phase Proteins 18114275 Chromatography Principles and Methods Principles and Methods 28909531 Hydrophobic Interaction 11001269 Purifying and Reversed Phase Challenging Proteins AVofl.fi 2n: Tiatgyg eCd hPrrooteminsatography CPrihncripolems aandt Moegthroadsphy Principles and Methods GE Healthcare Affinity Chromatography GLiEfe H Secaieltnhcceasre Imaging GLiEfe H Secaieltnhcceasre Size Exclusion Vol. 3: Specific Groups of Principles and Methods Chromatography B18io1m02o2le2c9ules LCaCseDrtraeenppsii312UUVV365 460473520532W 663305650685710IIRR785 29020301 P1r8i1n0ci2p2le1s8 and Methods Imaging Affinity Chromatography Principles and Methods SPriinzceip lEesx acnldu Mseiothnod sChromatography Vol. 3: Specific Groups of Biomolecules GLiEfe H Secaieltnhcceasre ÄKTA Laboratory-scale GE Healthcare Ion Exchange GLiEfe H Secaieltnhcceasre Spectrophotometry Chromatography Chromatography Handbook Systems Principles and Methods 29033182 Instrument Management 11000421 Handbook ÄICnsKhtrrTuoAmm™en tLa Mataobngaogrreaampteohntry Hy a-Snsydc bsaotoelkem s 29010831 ICPorihnncr iopElemxsc aahntdao Mngegtrhaeodp shy SHapndbeoocktrophotometry GLiEfe H Secaieltnhcceasre Biacore Assay GLiEfe H Secaieltnhcceasre Isolation of GLiEfe H Secaieltnhcceasre Strategies for Protein Handbook Mononuclear Cells Purification Biacore™ Assay Handbook 29019400 Methodology and Handbook Applications 28983331 Isolation of 18115269 mMethoodonlogoy annd auppclicalteionas r cells SPtrroatteeing iPeus rfiof irc ation Handbook GLiEfe H Secaieltnhcceasre Biacore Sensor Surface GLiEfe H Secaieltnhcceasre Microcarrier Cell Culture GLiEfe H Secaieltnhcceasre Western Blotting Biacore Handbook Principles and Methods Principles and Methods Sensor Surface Handbook BR100571 18114062 28999897 MCeicllr Cocualtrurrieer Western Blottin g Principles and Methods Principles and Methods GLiEfe H Secaieltnhcceasre Cell Separation Media GLiEfe H Secaieltnhcceasre Multimodal GLiEfe H Secaieltnhcceasre 2-D Electrophoresis using Methodology and Chromatography Immobilized pH Gradients Applications Handbook Principles and Methods 18111569 29054808 80642960 Cell Separation Media 2-D Electrophoresis Methodology and applications MChurlotimmaotdoagl raphy Principles and Methods Handbook imagination at work GLiEfe H Secaieltnhcceasre GST Gene Fusion System GLiEfe H Secaieltnhcceasre Nucleic Acid Sample Handbook Preparation for 18115758 Downstream Analyses Principles and Methods GFuSsTi oGne Snyes tem NPDrouewcpleanirscat rAteiocainmd fS oAarn m a plylsee s 28962400 Handbook Principles and Methods Affinity Chromatography Vol. 2: Tagged Proteins Contents Introduction ......................................................................................................................................9 Common acronyms and abbreviations ..........................................................................................................11 Symbols ..........................................................................................................................................................................12 Chapter 1 Expression and sample preparation ..........................................................................................13 Components of the expression system .........................................................................................................13 Choice of host ......................................................................................................................................................13 Choice of vector ..................................................................................................................................................14 Choice of tag ........................................................................................................................................................15 Sample preparation ..................................................................................................................................................18 Cell harvesting and extraction ....................................................................................................................18 Preparation for chromatographic purification ...................................................................................20 Protein stability ...................................................................................................................................................20 Sample clarification ..........................................................................................................................................21 Desalting and buffer exchange ..................................................................................................................22 Detection and quantitation ..........................................................................................................................22 Assessing protein expression ......................................................................................................................23 Chapter 2 Manual and automated purification ..........................................................................................25 Tagged recombinant proteins for simple purification .............................................................................25 Manual purification techniques ..........................................................................................................................25 Selection of purification equipment .................................................................................................................26 Chapter 3 Purification of histidine-tagged recombinant proteins ..........................................................29 Expression .....................................................................................................................................................................29 Purification overview ...............................................................................................................................................29 Imidazole ................................................................................................................................................................33 Cell lysis ...................................................................................................................................................................33 Sample preparation ..........................................................................................................................................34 Purification using precharged media .............................................................................................................35 Purification using Ni Sepharose products .....................................................................................................36 Purification using Ni Sepharose High Performance .................................................................................38 Column packing ..................................................................................................................................................38 Sample preparation ..........................................................................................................................................38 Buffer preparation .............................................................................................................................................39 Purification ............................................................................................................................................................39 Purification using Ni Sepharose 6 Fast Flow ................................................................................................40 Column packing ..................................................................................................................................................40 Sample preparation ..........................................................................................................................................41 Buffer preparation .............................................................................................................................................41 Purification ............................................................................................................................................................41 High-throughput screening using His MultiTrap HP and His MultiTrap FF 96-well filter plates ................................................................................................................42 Sample and buffer preparation ..................................................................................................................43 Centrifugation procedure for high-throughput screening............................................................43 Vacuum procedure for high-throughput screening .........................................................................44 Vacuum procedure ...........................................................................................................................................45 Application example.........................................................................................................................................45 Minipreps using His SpinTrap and His SpinTrap Kit ....................................................................................47 Cell lysis ...................................................................................................................................................................48 Sample and buffer preparation ..................................................................................................................48 2 18114275 AF Purification ............................................................................................................................................................48 Application example.........................................................................................................................................49 Purification using HisTrap HP and HisTrap FF ..............................................................................................49 Sample and buffer preparation ..................................................................................................................50 Purification ............................................................................................................................................................50 Application examples ......................................................................................................................................52 Purification from unclarified cell lysate using HisTrap FF crude .........................................................54 Sample and buffer preparation ..................................................................................................................55 Purification ............................................................................................................................................................55 Application examples ......................................................................................................................................56 Manual purification using HisTrap FF crude Kit with a syringe ...........................................................60 Sample preparation ..........................................................................................................................................60 Buffer preparation .............................................................................................................................................60 Standard purification .......................................................................................................................................62 Optimizing purification ....................................................................................................................................63 Application example.........................................................................................................................................64 Gravity-flow purification using His GraviTrap and His GraviTrap Kit ...............................................65 Sample and buffer preparation ..................................................................................................................66 Purification ............................................................................................................................................................66 Application example.........................................................................................................................................67 Condition screening for scaling up using HiScreen Ni FF ......................................................................68 Sample and buffer preparation ..................................................................................................................68 Purification ............................................................................................................................................................68 Application example.........................................................................................................................................69 Preparative purification using HisPrep FF 16/10 ........................................................................................70 Sample and buffer preparation ..................................................................................................................70 Purification ............................................................................................................................................................70 Application example.........................................................................................................................................71 Magnetic bead-based purification/screening using His Mag Sepharose Ni ...............................71 General magnetic separation steps .........................................................................................................72 Dispensing the medium slurry ....................................................................................................................72 Handling liquids ..................................................................................................................................................72 Incubation .............................................................................................................................................................72 Sample and buffer preparation ..................................................................................................................73 Purification ............................................................................................................................................................73 Application example.........................................................................................................................................74 Purification using Ni Sepharose excel products ........................................................................................75 Purification of histidine-tagged proteins secreted into eukaryotic cell culture supernatants using Ni Sepharose excel ................................................................................76 Column packing ..................................................................................................................................................76 Sample preparation .........................................................................................................................................77 Buffer preparation ...........................................................................................................................................77 Purification ............................................................................................................................................................78 Purification of histidine-tagged proteins secreted into eukaryotic cell culture supernatants using HisTrap excel ............................................................................................78 Sample and buffer preparation ..................................................................................................................79 Purification ...........................................................................................................................................................79 Application example.........................................................................................................................................79 Magnetic bead-based purification of histidine-tagged proteins secreted into eukaryotic cell culture supernatants using His Mag Sepharose excel ..................................80 General magnetic separation steps .........................................................................................................81 Dispensing the medium slurry ....................................................................................................................81 Handling liquids ..................................................................................................................................................81 Incubation .............................................................................................................................................................82 Sample and buffer preparation ..................................................................................................................82 Purification ...........................................................................................................................................................82 Application example.........................................................................................................................................83 18114275 AF 3 Purification using TALON Superflow products ...........................................................................................84 Purification using TALON Superflow .................................................................................................................84 Sample preparation ..........................................................................................................................................85 Buffer preparation ...........................................................................................................................................85 Column packing ..................................................................................................................................................85 Purification ............................................................................................................................................................86 High-throughput screening using His MultiTrap TALON .........................................................................87 Sample and buffer preparation ..................................................................................................................87 Purification ...........................................................................................................................................................87 Purification ...........................................................................................................................................................89 Rapid screening using His SpinTrap TALON ...................................................................................................90 Sample and buffer preparation ..................................................................................................................91 Purification ............................................................................................................................................................91 Manual purification using His GraviTrap TALON .........................................................................................92 Sample and buffer preparation ..................................................................................................................93 Purification ............................................................................................................................................................93 Application example.........................................................................................................................................93 Purification using HiTrap TALON crude ...........................................................................................................94 Sample and buffer preparation ..................................................................................................................94 Purification ............................................................................................................................................................94 Application example.........................................................................................................................................95 Purification using uncharged media ................................................................................................................96 Purification using IMAC Sepharose High Performance ..........................................................................99 Packing a column ..............................................................................................................................................99 Sample preparation ..........................................................................................................................................99 Buffer preparation ..........................................................................................................................................100 Charging the chromatography medium with metal ion .............................................................100 Purification ..........................................................................................................................................................101 Purification using IMAC Sepharose 6 Fast Flow .......................................................................................101 Packing a column ...........................................................................................................................................102 Sample preparation .......................................................................................................................................102 Buffer preparation ..........................................................................................................................................102 Charging the chromatography medium with metal ion .............................................................102 Purification .........................................................................................................................................................103 Purification using HiTrap IMAC HP and HiTrap IMAC FF .......................................................................103 Sample and buffer preparation ...............................................................................................................104 Charging the column with metal ion .....................................................................................................104 Purification .........................................................................................................................................................105 Application example......................................................................................................................................105 Condition screening for scaling up using HiScreen IMAC FF .............................................................107 Sample and buffer preparation ................................................................................................................107 Charging the chromatography medium with metal ions ............................................................107 Purification .........................................................................................................................................................108 Preparative purification using HiPrep IMAC FF 16/10 ...........................................................................109 Sample and buffer preparation ...............................................................................................................109 Charging the column with metal ion .....................................................................................................109 Purification ..........................................................................................................................................................110 Detection of histidine-tagged proteins ........................................................................................................111 Tag removal by enzymatic cleavage ............................................................................................................111 Application example......................................................................................................................................112 Troubleshooting ........................................................................................................................................................114 Chapter 4 Optimizing purification of histidine-tagged proteins ...........................................................117 Optimizing using imidazole .................................................................................................................................117 Optimizing using different metal ions ..........................................................................................................120 Optimizing using multistep purifications ....................................................................................................123 4 18114275 AF Chapter 5 Purification of GST-tagged recombinant proteins ................................................................125 Expression ..................................................................................................................................................................130 pGEX vectors .....................................................................................................................................................130 The host ...............................................................................................................................................................131 Insert DNA ..........................................................................................................................................................131 Optimizing expression ..................................................................................................................................132 Purification .................................................................................................................................................................132 General considerations for purification of GST-tagged proteins ....................................................133 Selecting equipment for purification ............................................................................................................134 Purification using Glutathione Sepharose High Performance, Glutathione Sepharose 4 Fast Flow, and Glutathione Sepharose 4B ...........................................135 Batch purification of GST-tagged proteins using Glutathione Sepharose HP, Glutathione Sepharose 4 FF, or Glutathione Sepharose 4B ......................................................136 Column purification of GST-tagged proteins using Glutathione Sepharose HP, Glutathione Sepharose 4 FF, or Glutathione Sepharose 4B .......................................................137 Column packing ...............................................................................................................................................137 Purification .........................................................................................................................................................137 High-throughput screening using GST MultiTrap 4B 96-well filter plates ..................................138 Sample preparation .......................................................................................................................................139 Buffer preparation ..........................................................................................................................................139 Centrifugation procedure for high-throughput screening.........................................................139 Centrifugation procedure ...........................................................................................................................140 Preparing the filter plate ..............................................................................................................................141 Minipreps using GST SpinTrap ...........................................................................................................................142 Sample preparation .......................................................................................................................................142 Buffer preparation ..........................................................................................................................................142 Purification ..........................................................................................................................................................143 Gravity-flow purification using GST GraviTrap ..........................................................................................143 Sample preparation .......................................................................................................................................144 Buffer preparation ..........................................................................................................................................144 Purification .........................................................................................................................................................144 Purification using GSTrap HP, GSTrap FF, and GSTrap 4B columns..................................................145 Sample preparation .......................................................................................................................................146 Buffer preparation ..........................................................................................................................................146 Purification ..........................................................................................................................................................147 Application examples ...................................................................................................................................148 Preparative purification using GSTPrep FF 16/10 column ...................................................................151 Sample preparation ........................................................................................................................................151 Buffer preparation ...........................................................................................................................................151 Purification .........................................................................................................................................................152 Application example......................................................................................................................................152 Troubleshooting of purification methods ...................................................................................................155 Detection of GST-tagged proteins ..................................................................................................................159 GST Detection Module with CDNB enzymatic assay.....................................................................159 Western blot ......................................................................................................................................................160 Troubleshooting of detection methods ........................................................................................................161 Removal of GST tag by enzymatic cleavage .............................................................................................162 Cleavage and purification of GST-tagged protein bound to GSTrap FF ...............................166 Application examples ...................................................................................................................................168 1. Purification of human hippocalcin using GSTrap FF columns in series with on-column cleavage by PreScission Protease .................................................................................168 Cleavage and purification of GST-tagged protein eluted from GSTrap FF ..........................172 Cleavage and purification of GST-tagged protein bound to Glutathione Sepharose in batch mode .................................................................................................173 Removal of thrombin and Factor Xa using HiTrap Benzamidine FF (high sub) .................175 Troubleshooting of cleavage methods .........................................................................................................176 18114275 AF 5 Chapter 6 Purification of MBP-tagged recombinant proteins ...............................................................178 Purification using Dextrin Sepharose High Performance .....................................................................178 Column packing ................................................................................................................................................179 Sample preparation ........................................................................................................................................179 Buffer preparation ...........................................................................................................................................179 Purification ..........................................................................................................................................................179 Purification using MBPTrap HP columns ......................................................................................................180 Sample preparation .......................................................................................................................................180 Buffer preparation ..........................................................................................................................................180 Purification .........................................................................................................................................................181 Application examples ...................................................................................................................................182 1. Automated two-step purification on ÄKTAxpress .....................................................................182 2. Purification of a protein involved in metabolic disease ..........................................................183 3. Scaling up ......................................................................................................................................................184 Chapter 7 Purification of Strep-tag II recombinant proteins .................................................................187 Purification using StrepTactin Sepharose High Performance ............................................................187 Column packing ...............................................................................................................................................188 Sample preparation .......................................................................................................................................188 Buffer preparation ..........................................................................................................................................188 Purification .........................................................................................................................................................188 Purification using StrepTrap HP 1 ml and 5 ml ..........................................................................................189 Sample preparation .......................................................................................................................................189 Buffer preparation ..........................................................................................................................................189 Purification .........................................................................................................................................................190 Application examples ...................................................................................................................................190 Individual HisTrap HP purification ...........................................................................................................191 Individual StrepTrap HP purification ......................................................................................................191 Two-step HisTrap HP and StrepTrap HP purification .....................................................................192 Chapter 8 Purification of protein A-tagged proteins ...............................................................................197 Performing a separation using IgG Sepharose 6 Fast Flow ................................................................197 Buffer preparation ...........................................................................................................................................197 Application example......................................................................................................................................198 Chemical stability ...........................................................................................................................................198 Storage .................................................................................................................................................................198 Chapter 9 Affinity chromatography in a purification strategy (CIPP) ....................................................199 Applying CIPP ..............................................................................................................................................................200 Selection and combination of purification techniques..........................................................................200 Chapter 10 Handling inclusion bodies ..........................................................................................................205 Optimizing for soluble expression ..........................................................................................................205 Solubilizing inclusion bodies ......................................................................................................................206 Refolding of solubilized recombinant proteins .................................................................................207 Analysis of correctly folded proteins .....................................................................................................208 Application .........................................................................................................................................................209 Screening conditions for refolding using IMAC ................................................................................211 6 18114275 AF Chapter 11 Desalting/buffer exchange and concentration .....................................................................213 General considerations ........................................................................................................................................217 Desalting larger sample volumes using HiTrap and HiPrep columns ...................................217 Buffer preparation ...........................................................................................................................................217 Sample preparation ........................................................................................................................................217 Buffer exchange ..............................................................................................................................................218 Small-scale desalting and buffer exchange with PD desalting columns ......................................218 PD SpinTrap G-25 .............................................................................................................................................218 PD MultiTrap G-25 ...........................................................................................................................................219 PD MiniTrap G-25 and PD MidiTrap G-25 .............................................................................................221 PD-10 Desalting columns ............................................................................................................................223 PD MiniTrap G-10 and PD MidiTrap G-10 ..............................................................................................225 HiTrap Desalting columns ..................................................................................................................................226 Scaling up desalting from HiTrap to HiPrep Desalting .........................................................................229 Protein sample concentration ..........................................................................................................................229 Appendix 1 Characteristics of Ni Sepharose, Ni Sepharose excel, TALON Superflow, and uncharged IMAC Sepharose products .....................................................................................231 Ni Sepharose products .........................................................................................................................................231 Stripping, recharging, and cleaning of Ni Sepharose products .......................................................237 Stripping and recharging ............................................................................................................................237 Cleaning-in-place ...........................................................................................................................................237 To remove ionically bound protein: ........................................................................................................237 To remove precipitated proteins, hydrophobically bound proteins, and lipoproteins: .237 To remove hydrophobically bound proteins, lipoproteins, and lipids: ..................................237 Ni Sepharose excel/His Mag Sepharose excel products .....................................................................238 Cleaning of Ni Sepharose excel products ...................................................................................................239 Cleaning-in-place ...........................................................................................................................................239 To remove ionically bound proteins: ......................................................................................................239 To remove precipitated proteins, hydrophobically bound proteins, and lipoproteins: .239 To remove hydrophobically bound proteins, lipoproteins, and lipids: ..................................239 TALON Superflow products ................................................................................................................................240 Stripping, recharging, and cleaning of TALON Superflow products ..............................................242 Stripping and recharging ............................................................................................................................242 Uncharged IMAC Sepharose products ..........................................................................................................243 Stripping, recharging, and cleaning of IMAC Sepharose products .................................................247 Stripping and recharging .............................................................................................................................247 Cleaning-in-place ...........................................................................................................................................248 To remove ionically bound protein: ........................................................................................................248 To remove precipitated proteins, hydrophobically bound proteins, and lipoproteins: .....248 To remove hydrophobically bound proteins, lipoproteins, and lipids: ..................................248 Appendix 2 Characteristics of Glutathione Sepharose products .............................................................249 Appendix 3 Characteristics of Dextrin Sepharose High Performance products ...................................253 Cleaning of Dextrin Sepharose products ....................................................................................................254 18114275 AF 7 Appendix 4 Characteristics of StrepTactin Sepharose High Performance products ...........................255 Regeneration and cleaning of StrepTactin Sepharose High Performance ................................257 Regeneration and cleaning of StrepTrap HP 1 ml and 5 ml ...............................................................257 Appendix 5 Precipitation and resolubilization ............................................................................................258 Fractional precipitation .......................................................................................................................................258 Ammonium sulfate precipitation ....................................................................................................................259 Solutions needed for precipitation: ........................................................................................................260 Appendix 6 Column packing and preparation ............................................................................................262 Column packing and efficiency .......................................................................................................................264 Custom column packing .............................................................................................................................265 Appendix 7 Conversion data ...........................................................................................................................266 Proteins ........................................................................................................................................................................266 Column pressures ...................................................................................................................................................266 Appendix 8 Converting from flow velocity to volumetric flow rates .......................................................267 From flow velocity (cm/h) to volumetric flow rate (ml/min) ..............................................................267 From volumetric flow rate (ml/min) to flow velocity (cm/h) ................................................................267 From volumetric flow rate (ml/min) to using a syringe ........................................................................267 Appendix 9 GST vectors ...................................................................................................................................268 Appendix 10 Table of amino acids ...................................................................................................................270 Appendix 11 Principles and standard conditions for different purification techniques ........................272 Affinity chromatography (AC)............................................................................................................................272 Ion exchange chromatography (IEX) .............................................................................................................273 Method development (in priority order) ................................................................................................274 Hydrophobic interaction chromatography (HIC) ......................................................................................274 Method development (in priority order) ................................................................................................275 Size exclusion chromatography (SEC) ............................................................................................................276 Reversed phase chromatography (RPC) ......................................................................................................277 Method development ....................................................................................................................................277 Appendix 12 Tables for Vivaspin sample concentrators ............................................................................278 Product index ................................................................................................................................280 Related literature ........................................................................................................................282 Ordering information ..................................................................................................................284 8 18114275 AF

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Affinity Chromatography. Vol. 2: Tagged Proteins. 18114275. Affinity Chromatography. Vol. 3: Specific Groups of Biomolecules. GE Healthcare. Affinity
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GE Healthcare A f fi n it y C h r o m a t o g r a p h y – V o l. 2 : T a g g e d P r o t e in s www.gelifesciences.com GE, GE monogram, Amersham, ÄKTA, Biacore, Capto, ECL, ECL Plex, ECL Select, ExcelGel, GraviTrap, GSTPrep, GSTrap, HiLoad, HiPrep, HiScale, HiScreen, HisPrep, HisTrap, HiTrap, Hybond, ImageQuant, MabSelect, MabSelect SuRe, MabSelect Xtra, MBPTrap, MidiTrap, MiniTrap, Mono Q, Multiphor, MultiTrap, PhastGel, PhastSystem, PreScission, PrimeView, Protran, Rainbow, RESOURCE, Sephacryl, Sephadex, Sepharose, SpinTrap, SOURCE, StrepTrap, Superdex, Superloop, Tricorn, and UNICORN are trademarks of General Electric Company. AcTEV, GIBCO, High Five, Novex, and SimplyBlue are trademarks of Life Technologies Corp. Amicon, Benzonase, BugBuster, Diaflo, and Milli-Q are trademarks of Merck KGaA. BCA and HisProbe are trademarks of Thermo Fisher Scientific. Coomassie is a trademark of Imperial Chemical Industries, Ltd. CYMAL and SAFC are trademarks of Affymetrix Inc. Eppendorf is a trademark of Eppendorf AG. EX-CELL and SAFC are trademarks of Sigma-Aldrich Co. Gel Doc is a trademark of BioRad Laboratories Inc. GenBank is a trademark of US Dept. of Health & Human Services. Kathon is a trademark of Dow Chemical Co. MODDE is a trademark of Umetrics AB. Pefabloc is a trademark of Pentapharm Ltd. Strep-Tactin and Strep-tag are trademarks of IBA GmbH. Superflow is a trademark of Sterogene Bioseparations Inc. TALON is a registered trademark of Clontech Laboratories Inc. Tween is a trademark of Uniqema Americas LLC. Vibracell is a trademark of Sonics & Materials, Inc. Vivaspin is a trademark of Sartorius Stedim Biotech GmbH. ECL Prime: This product or portions thereof is manufactured and sold under license from Cyanagen Srl and is subject Affinity Chromatography of US patent 7,855,287, US Patent 7,803,573, and US Patent 9,040,252, together with other equivalent granted patents and patent applications in other countries. IMAC Sepharose products, Ni Sepharose products and Fe Sepharose products: These products are sold under a license from Sigma-Aldrich under patent number EP 1276716 (Metal chelating compositions) and equivalent patents and Vol. 2: Tagged Proteins patent applications in other countries. StrepTrap HP and StrepTactin Sepharose High Performance: These products are covered by US patent number GE Healthcare Bio-Sciences AB 6,103,493 and equivalent patents and patent applications in other countries. The purchase of StrepTrap HP and StrepTactin Sepharose High Performance includes a license under such patents for nonprofit and in-house research Björkgatan 30 only. Please contact IBA ([email protected]) for further information on licenses for commercial use of StrepTactin. 751 84 Uppsala TALON Superflow and HiTrap TALON: TALON is a registered trademark of Clontech Laboratories Inc. © 2000–2016 General Electric Company. First published Dec 2000. Sweden All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information GE Healthcare UK Ltd, Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA, UK GE Healthcare Bio-Sciences Corp. 100 Results Way, Marlborough, MA 01752, USA GE Healthcare Dharmacon, Inc., 2650 Crescent Dr., Lafayette, CO 80026, USA imagination atH ywClonoe Larbkoratories, Inc., 925 W 1800 S, Logan, UT 84321, USA imagination at work GE Healthcare Europe GmbH, Munzinger Strasse 5, D-79111 Freiburg, Germany GE Healthcare Japan Corporation, Sanken Bldg. 3-25-1, Hyakunincho, Shinjuku-ku, Tokyo 169-0073, Japan For local office contact information, visit www.gelifesciences.com/contact imagination at work 18114275 AF 04/2016 Handbooks from GE Healthcare Life Sciences For more information refer to www.gelifesciences.com/handbooks GE Healthcare Affinity Chromatography GLiEfe H Secaieltnhcceasre High-throughput Process GLiEfe H Secaieltnhcceasre Protein Sample Vol. 1: Antibodies Development with Preparation 18103746 HPwriiogthch e-Ptsrhser DDouiecgvtoehrlpo™up Ptmlaetenst PreDictor Plates Handbook Principles and Methods Principles and Methods 28988741 28940358 Protein Sample AVofl.fi 1n: Ainttyib oCdhiesromatography PHarnedbpooakration GE Healthcare Affinity Chromatography GLiEfe H Secaieltnhcceasre Hydrophobic Interaction GLiEfe H Secaieltnhcceasre Purifying Challenging Vol. 2: Tagged Proteins and Reversed Phase Proteins 18114275 Chromatography Principles and Methods Principles and Methods 28909531 Hydrophobic Interaction 11001269 Purifying and Reversed Phase Challenging Proteins AVofl.fi 2n: Tiatgyg eCd hPrrooteminsatography CPrihncripolems aandt Moegthroadsphy Principles and Methods GE Healthcare Affinity Chromatography GLiEfe H Secaieltnhcceasre Imaging GLiEfe H Secaieltnhcceasre Size Exclusion Vol. 3: Specific Groups of Principles and Methods Chromatography B18io1m02o2le2c9ules LCaCseDrtraeenppsii312UUVV365 460473520532W 663305650685710IIRR785 29020301 P1r8i1n0ci2p2le1s8 and Methods Imaging Affinity Chromatography Principles and Methods SPriinzceip lEesx acnldu Mseiothnod sChromatography Vol. 3: Specific Groups of Biomolecules GLiEfe H Secaieltnhcceasre ÄKTA Laboratory-scale GE Healthcare Ion Exchange GLiEfe H Secaieltnhcceasre Spectrophotometry Chromatography Chromatography Handbook Systems Principles and Methods 29033182 Instrument Management 11000421 Handbook ÄICnsKhtrrTuoAmm™en tLa Mataobngaogrreaampteohntry Hy a-Snsydc bsaotoelkem s 29010831 ICPorihnncr iopElemxsc aahntdao Mngegtrhaeodp shy SHapndbeoocktrophotometry GLiEfe H Secaieltnhcceasre Biacore Assay GLiEfe H Secaieltnhcceasre Isolation of GLiEfe H Secaieltnhcceasre Strategies for Protein Handbook Mononuclear Cells Purification Biacore™ Assay Handbook 29019400 Methodology and Handbook Applications 28983331 Isolation of 18115269 mMethoodonlogoy annd auppclicalteionas r cells SPtrroatteeing iPeus rfiof irc ation Handbook GLiEfe H Secaieltnhcceasre Biacore Sensor Surface GLiEfe H Secaieltnhcceasre Microcarrier Cell Culture GLiEfe H Secaieltnhcceasre Western Blotting Biacore Handbook Principles and Methods Principles and Methods Sensor Surface Handbook BR100571 18114062 28999897 MCeicllr Cocualtrurrieer Western Blottin g Principles and Methods Principles and Methods GLiEfe H Secaieltnhcceasre Cell Separation Media GLiEfe H Secaieltnhcceasre Multimodal GLiEfe H Secaieltnhcceasre 2-D Electrophoresis using Methodology and Chromatography Immobilized pH Gradients Applications Handbook Principles and Methods 18111569 29054808 80642960 Cell Separation Media 2-D Electrophoresis Methodology and applications MChurlotimmaotdoagl raphy Principles and Methods Handbook imagination at work GLiEfe H Secaieltnhcceasre GST Gene Fusion System GLiEfe H Secaieltnhcceasre Nucleic Acid Sample Handbook Preparation for 18115758 Downstream Analyses Principles and Methods GFuSsTi oGne Snyes tem NPDrouewcpleanirscat rAteiocainmd fS oAarn m a plylsee s 28962400 Handbook Principles and Methods Affinity Chromatography Vol. 2: Tagged Proteins Contents Introduction ......................................................................................................................................9 Common acronyms and abbreviations ..........................................................................................................11 Symbols ..........................................................................................................................................................................12 Chapter 1 Expression and sample preparation ..........................................................................................13 Components of the expression system .........................................................................................................13 Choice of host ......................................................................................................................................................13 Choice of vector ..................................................................................................................................................14 Choice of tag ........................................................................................................................................................15 Sample preparation ..................................................................................................................................................18 Cell harvesting and extraction ....................................................................................................................18 Preparation for chromatographic purification ...................................................................................20 Protein stability ...................................................................................................................................................20 Sample clarification ..........................................................................................................................................21 Desalting and buffer exchange ..................................................................................................................22 Detection and quantitation ..........................................................................................................................22 Assessing protein expression ......................................................................................................................23 Chapter 2 Manual and automated purification ..........................................................................................25 Tagged recombinant proteins for simple purification .............................................................................25 Manual purification techniques ..........................................................................................................................25 Selection of purification equipment .................................................................................................................26 Chapter 3 Purification of histidine-tagged recombinant proteins ..........................................................29 Expression .....................................................................................................................................................................29 Purification overview ...............................................................................................................................................29 Imidazole ................................................................................................................................................................33 Cell lysis ...................................................................................................................................................................33 Sample preparation ..........................................................................................................................................34 Purification using precharged media .............................................................................................................35 Purification using Ni Sepharose products .....................................................................................................36 Purification using Ni Sepharose High Performance .................................................................................38 Column packing ..................................................................................................................................................38 Sample preparation ..........................................................................................................................................38 Buffer preparation .............................................................................................................................................39 Purification ............................................................................................................................................................39 Purification using Ni Sepharose 6 Fast Flow ................................................................................................40 Column packing ..................................................................................................................................................40 Sample preparation ..........................................................................................................................................41 Buffer preparation .............................................................................................................................................41 Purification ............................................................................................................................................................41 High-throughput screening using His MultiTrap HP and His MultiTrap FF 96-well filter plates ................................................................................................................42 Sample and buffer preparation ..................................................................................................................43 Centrifugation procedure for high-throughput screening............................................................43 Vacuum procedure for high-throughput screening .........................................................................44 Vacuum procedure ...........................................................................................................................................45 Application example.........................................................................................................................................45 Minipreps using His SpinTrap and His SpinTrap Kit ....................................................................................47 Cell lysis ...................................................................................................................................................................48 Sample and buffer preparation ..................................................................................................................48 2 18114275 AF Purification ............................................................................................................................................................48 Application example.........................................................................................................................................49 Purification using HisTrap HP and HisTrap FF ..............................................................................................49 Sample and buffer preparation ..................................................................................................................50 Purification ............................................................................................................................................................50 Application examples ......................................................................................................................................52 Purification from unclarified cell lysate using HisTrap FF crude .........................................................54 Sample and buffer preparation ..................................................................................................................55 Purification ............................................................................................................................................................55 Application examples ......................................................................................................................................56 Manual purification using HisTrap FF crude Kit with a syringe ...........................................................60 Sample preparation ..........................................................................................................................................60 Buffer preparation .............................................................................................................................................60 Standard purification .......................................................................................................................................62 Optimizing purification ....................................................................................................................................63 Application example.........................................................................................................................................64 Gravity-flow purification using His GraviTrap and His GraviTrap Kit ...............................................65 Sample and buffer preparation ..................................................................................................................66 Purification ............................................................................................................................................................66 Application example.........................................................................................................................................67 Condition screening for scaling up using HiScreen Ni FF ......................................................................68 Sample and buffer preparation ..................................................................................................................68 Purification ............................................................................................................................................................68 Application example.........................................................................................................................................69 Preparative purification using HisPrep FF 16/10 ........................................................................................70 Sample and buffer preparation ..................................................................................................................70 Purification ............................................................................................................................................................70 Application example.........................................................................................................................................71 Magnetic bead-based purification/screening using His Mag Sepharose Ni ...............................71 General magnetic separation steps .........................................................................................................72 Dispensing the medium slurry ....................................................................................................................72 Handling liquids ..................................................................................................................................................72 Incubation .............................................................................................................................................................72 Sample and buffer preparation ..................................................................................................................73 Purification ............................................................................................................................................................73 Application example.........................................................................................................................................74 Purification using Ni Sepharose excel products ........................................................................................75 Purification of histidine-tagged proteins secreted into eukaryotic cell culture supernatants using Ni Sepharose excel ................................................................................76 Column packing ..................................................................................................................................................76 Sample preparation .........................................................................................................................................77 Buffer preparation ...........................................................................................................................................77 Purification ............................................................................................................................................................78 Purification of histidine-tagged proteins secreted into eukaryotic cell culture supernatants using HisTrap excel ............................................................................................78 Sample and buffer preparation ..................................................................................................................79 Purification ...........................................................................................................................................................79 Application example.........................................................................................................................................79 Magnetic bead-based purification of histidine-tagged proteins secreted into eukaryotic cell culture supernatants using His Mag Sepharose excel ..................................80 General magnetic separation steps .........................................................................................................81 Dispensing the medium slurry ....................................................................................................................81 Handling liquids ..................................................................................................................................................81 Incubation .............................................................................................................................................................82 Sample and buffer preparation ..................................................................................................................82 Purification ...........................................................................................................................................................82 Application example.........................................................................................................................................83 18114275 AF 3 Purification using TALON Superflow products ...........................................................................................84 Purification using TALON Superflow .................................................................................................................84 Sample preparation ..........................................................................................................................................85 Buffer preparation ...........................................................................................................................................85 Column packing ..................................................................................................................................................85 Purification ............................................................................................................................................................86 High-throughput screening using His MultiTrap TALON .........................................................................87 Sample and buffer preparation ..................................................................................................................87 Purification ...........................................................................................................................................................87 Purification ...........................................................................................................................................................89 Rapid screening using His SpinTrap TALON ...................................................................................................90 Sample and buffer preparation ..................................................................................................................91 Purification ............................................................................................................................................................91 Manual purification using His GraviTrap TALON .........................................................................................92 Sample and buffer preparation ..................................................................................................................93 Purification ............................................................................................................................................................93 Application example.........................................................................................................................................93 Purification using HiTrap TALON crude ...........................................................................................................94 Sample and buffer preparation ..................................................................................................................94 Purification ............................................................................................................................................................94 Application example.........................................................................................................................................95 Purification using uncharged media ................................................................................................................96 Purification using IMAC Sepharose High Performance ..........................................................................99 Packing a column ..............................................................................................................................................99 Sample preparation ..........................................................................................................................................99 Buffer preparation ..........................................................................................................................................100 Charging the chromatography medium with metal ion .............................................................100 Purification ..........................................................................................................................................................101 Purification using IMAC Sepharose 6 Fast Flow .......................................................................................101 Packing a column ...........................................................................................................................................102 Sample preparation .......................................................................................................................................102 Buffer preparation ..........................................................................................................................................102 Charging the chromatography medium with metal ion .............................................................102 Purification .........................................................................................................................................................103 Purification using HiTrap IMAC HP and HiTrap IMAC FF .......................................................................103 Sample and buffer preparation ...............................................................................................................104 Charging the column with metal ion .....................................................................................................104 Purification .........................................................................................................................................................105 Application example......................................................................................................................................105 Condition screening for scaling up using HiScreen IMAC FF .............................................................107 Sample and buffer preparation ................................................................................................................107 Charging the chromatography medium with metal ions ............................................................107 Purification .........................................................................................................................................................108 Preparative purification using HiPrep IMAC FF 16/10 ...........................................................................109 Sample and buffer preparation ...............................................................................................................109 Charging the column with metal ion .....................................................................................................109 Purification ..........................................................................................................................................................110 Detection of histidine-tagged proteins ........................................................................................................111 Tag removal by enzymatic cleavage ............................................................................................................111 Application example......................................................................................................................................112 Troubleshooting ........................................................................................................................................................114 Chapter 4 Optimizing purification of histidine-tagged proteins ...........................................................117 Optimizing using imidazole .................................................................................................................................117 Optimizing using different metal ions ..........................................................................................................120 Optimizing using multistep purifications ....................................................................................................123 4 18114275 AF Chapter 5 Purification of GST-tagged recombinant proteins ................................................................125 Expression ..................................................................................................................................................................130 pGEX vectors .....................................................................................................................................................130 The host ...............................................................................................................................................................131 Insert DNA ..........................................................................................................................................................131 Optimizing expression ..................................................................................................................................132 Purification .................................................................................................................................................................132 General considerations for purification of GST-tagged proteins ....................................................133 Selecting equipment for purification ............................................................................................................134 Purification using Glutathione Sepharose High Performance, Glutathione Sepharose 4 Fast Flow, and Glutathione Sepharose 4B ...........................................135 Batch purification of GST-tagged proteins using Glutathione Sepharose HP, Glutathione Sepharose 4 FF, or Glutathione Sepharose 4B ......................................................136 Column purification of GST-tagged proteins using Glutathione Sepharose HP, Glutathione Sepharose 4 FF, or Glutathione Sepharose 4B .......................................................137 Column packing ...............................................................................................................................................137 Purification .........................................................................................................................................................137 High-throughput screening using GST MultiTrap 4B 96-well filter plates ..................................138 Sample preparation .......................................................................................................................................139 Buffer preparation ..........................................................................................................................................139 Centrifugation procedure for high-throughput screening.........................................................139 Centrifugation procedure ...........................................................................................................................140 Preparing the filter plate ..............................................................................................................................141 Minipreps using GST SpinTrap ...........................................................................................................................142 Sample preparation .......................................................................................................................................142 Buffer preparation ..........................................................................................................................................142 Purification ..........................................................................................................................................................143 Gravity-flow purification using GST GraviTrap ..........................................................................................143 Sample preparation .......................................................................................................................................144 Buffer preparation ..........................................................................................................................................144 Purification .........................................................................................................................................................144 Purification using GSTrap HP, GSTrap FF, and GSTrap 4B columns..................................................145 Sample preparation .......................................................................................................................................146 Buffer preparation ..........................................................................................................................................146 Purification ..........................................................................................................................................................147 Application examples ...................................................................................................................................148 Preparative purification using GSTPrep FF 16/10 column ...................................................................151 Sample preparation ........................................................................................................................................151 Buffer preparation ...........................................................................................................................................151 Purification .........................................................................................................................................................152 Application example......................................................................................................................................152 Troubleshooting of purification methods ...................................................................................................155 Detection of GST-tagged proteins ..................................................................................................................159 GST Detection Module with CDNB enzymatic assay.....................................................................159 Western blot ......................................................................................................................................................160 Troubleshooting of detection methods ........................................................................................................161 Removal of GST tag by enzymatic cleavage .............................................................................................162 Cleavage and purification of GST-tagged protein bound to GSTrap FF ...............................166 Application examples ...................................................................................................................................168 1. Purification of human hippocalcin using GSTrap FF columns in series with on-column cleavage by PreScission Protease .................................................................................168 Cleavage and purification of GST-tagged protein eluted from GSTrap FF ..........................172 Cleavage and purification of GST-tagged protein bound to Glutathione Sepharose in batch mode .................................................................................................173 Removal of thrombin and Factor Xa using HiTrap Benzamidine FF (high sub) .................175 Troubleshooting of cleavage methods .........................................................................................................176 18114275 AF 5 Chapter 6 Purification of MBP-tagged recombinant proteins ...............................................................178 Purification using Dextrin Sepharose High Performance .....................................................................178 Column packing ................................................................................................................................................179 Sample preparation ........................................................................................................................................179 Buffer preparation ...........................................................................................................................................179 Purification ..........................................................................................................................................................179 Purification using MBPTrap HP columns ......................................................................................................180 Sample preparation .......................................................................................................................................180 Buffer preparation ..........................................................................................................................................180 Purification .........................................................................................................................................................181 Application examples ...................................................................................................................................182 1. Automated two-step purification on ÄKTAxpress .....................................................................182 2. Purification of a protein involved in metabolic disease ..........................................................183 3. Scaling up ......................................................................................................................................................184 Chapter 7 Purification of Strep-tag II recombinant proteins .................................................................187 Purification using StrepTactin Sepharose High Performance ............................................................187 Column packing ...............................................................................................................................................188 Sample preparation .......................................................................................................................................188 Buffer preparation ..........................................................................................................................................188 Purification .........................................................................................................................................................188 Purification using StrepTrap HP 1 ml and 5 ml ..........................................................................................189 Sample preparation .......................................................................................................................................189 Buffer preparation ..........................................................................................................................................189 Purification .........................................................................................................................................................190 Application examples ...................................................................................................................................190 Individual HisTrap HP purification ...........................................................................................................191 Individual StrepTrap HP purification ......................................................................................................191 Two-step HisTrap HP and StrepTrap HP purification .....................................................................192 Chapter 8 Purification of protein A-tagged proteins ...............................................................................197 Performing a separation using IgG Sepharose 6 Fast Flow ................................................................197 Buffer preparation ...........................................................................................................................................197 Application example......................................................................................................................................198 Chemical stability ...........................................................................................................................................198 Storage .................................................................................................................................................................198 Chapter 9 Affinity chromatography in a purification strategy (CIPP) ....................................................199 Applying CIPP ..............................................................................................................................................................200 Selection and combination of purification techniques..........................................................................200 Chapter 10 Handling inclusion bodies ..........................................................................................................205 Optimizing for soluble expression ..........................................................................................................205 Solubilizing inclusion bodies ......................................................................................................................206 Refolding of solubilized recombinant proteins .................................................................................207 Analysis of correctly folded proteins .....................................................................................................208 Application .........................................................................................................................................................209 Screening conditions for refolding using IMAC ................................................................................211 6 18114275 AF Chapter 11 Desalting/buffer exchange and concentration .....................................................................213 General considerations ........................................................................................................................................217 Desalting larger sample volumes using HiTrap and HiPrep columns ...................................217 Buffer preparation ...........................................................................................................................................217 Sample preparation ........................................................................................................................................217 Buffer exchange ..............................................................................................................................................218 Small-scale desalting and buffer exchange with PD desalting columns ......................................218 PD SpinTrap G-25 .............................................................................................................................................218 PD MultiTrap G-25 ...........................................................................................................................................219 PD MiniTrap G-25 and PD MidiTrap G-25 .............................................................................................221 PD-10 Desalting columns ............................................................................................................................223 PD MiniTrap G-10 and PD MidiTrap G-10 ..............................................................................................225 HiTrap Desalting columns ..................................................................................................................................226 Scaling up desalting from HiTrap to HiPrep Desalting .........................................................................229 Protein sample concentration ..........................................................................................................................229 Appendix 1 Characteristics of Ni Sepharose, Ni Sepharose excel, TALON Superflow, and uncharged IMAC Sepharose products .....................................................................................231 Ni Sepharose products .........................................................................................................................................231 Stripping, recharging, and cleaning of Ni Sepharose products .......................................................237 Stripping and recharging ............................................................................................................................237 Cleaning-in-place ...........................................................................................................................................237 To remove ionically bound protein: ........................................................................................................237 To remove precipitated proteins, hydrophobically bound proteins, and lipoproteins: .237 To remove hydrophobically bound proteins, lipoproteins, and lipids: ..................................237 Ni Sepharose excel/His Mag Sepharose excel products .....................................................................238 Cleaning of Ni Sepharose excel products ...................................................................................................239 Cleaning-in-place ...........................................................................................................................................239 To remove ionically bound proteins: ......................................................................................................239 To remove precipitated proteins, hydrophobically bound proteins, and lipoproteins: .239 To remove hydrophobically bound proteins, lipoproteins, and lipids: ..................................239 TALON Superflow products ................................................................................................................................240 Stripping, recharging, and cleaning of TALON Superflow products ..............................................242 Stripping and recharging ............................................................................................................................242 Uncharged IMAC Sepharose products ..........................................................................................................243 Stripping, recharging, and cleaning of IMAC Sepharose products .................................................247 Stripping and recharging .............................................................................................................................247 Cleaning-in-place ...........................................................................................................................................248 To remove ionically bound protein: ........................................................................................................248 To remove precipitated proteins, hydrophobically bound proteins, and lipoproteins: .....248 To remove hydrophobically bound proteins, lipoproteins, and lipids: ..................................248 Appendix 2 Characteristics of Glutathione Sepharose products .............................................................249 Appendix 3 Characteristics of Dextrin Sepharose High Performance products ...................................253 Cleaning of Dextrin Sepharose products ....................................................................................................254 18114275 AF 7 Appendix 4 Characteristics of StrepTactin Sepharose High Performance products ...........................255 Regeneration and cleaning of StrepTactin Sepharose High Performance ................................257 Regeneration and cleaning of StrepTrap HP 1 ml and 5 ml ...............................................................257 Appendix 5 Precipitation and resolubilization ............................................................................................258 Fractional precipitation .......................................................................................................................................258 Ammonium sulfate precipitation ....................................................................................................................259 Solutions needed for precipitation: ........................................................................................................260 Appendix 6 Column packing and preparation ............................................................................................262 Column packing and efficiency .......................................................................................................................264 Custom column packing .............................................................................................................................265 Appendix 7 Conversion data ...........................................................................................................................266 Proteins ........................................................................................................................................................................266 Column pressures ...................................................................................................................................................266 Appendix 8 Converting from flow velocity to volumetric flow rates .......................................................267 From flow velocity (cm/h) to volumetric flow rate (ml/min) ..............................................................267 From volumetric flow rate (ml/min) to flow velocity (cm/h) ................................................................267 From volumetric flow rate (ml/min) to using a syringe ........................................................................267 Appendix 9 GST vectors ...................................................................................................................................268 Appendix 10 Table of amino acids ...................................................................................................................270 Appendix 11 Principles and standard conditions for different purification techniques ........................272 Affinity chromatography (AC)............................................................................................................................272 Ion exchange chromatography (IEX) .............................................................................................................273 Method development (in priority order) ................................................................................................274 Hydrophobic interaction chromatography (HIC) ......................................................................................274 Method development (in priority order) ................................................................................................275 Size exclusion chromatography (SEC) ............................................................................................................276 Reversed phase chromatography (RPC) ......................................................................................................277 Method development ....................................................................................................................................277 Appendix 12 Tables for Vivaspin sample concentrators ............................................................................278 Product index ................................................................................................................................280 Related literature ........................................................................................................................282 Ordering information ..................................................................................................................284 8 18114275 AF

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Affinity Chromatography. Vol. 2: Tagged Proteins. 18114275. Affinity Chromatography. Vol. 3: Specific Groups of Biomolecules. GE Healthcare. Affinity
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