Lumi-Imager™ F1: Lab Protocols

57 Pages·1998·4.666 MB·English

by  Onno Bakker Ph.D. (auth.)

Onno Bakker Lumi-lmagerfM.... "TI Fl - Lab Protocols
Springer-Verlag Berlin Heidelberg GmbH
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ONNO BAKKER, Ph.D.
Academic Medical Centre
Endocrinology, FS-l71
Meibergdreef 9
110S AZ Amsterdam
Netherlands
ISBN 978-3-540-64794-2 ISBN 978-3-662-08431-1 (eBook)
DOI 10.1007/978-3-662-08431-1
Library of Congress Cataloging-in-Publication Data applied for.
Die Deutsche Bibliothek - CIP-Einheitsaufnahme
Bakker, Onno: Lumi-imager F1 - lab protocols/Onno Bakker (author). - Berlin; Hei
delberg, New York; Barcelona; Budapest; Hong Kong; London; Mailand; Paris; Singa
pore; Tokyo: Springer, 1998
This work is subject to copyright. All rights are reserved, whether the whole or part
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Verlag. Violations are liable for prosecution under the German Copyright Law.
© Springer-Verlag Berlin Heidelberg 1998
Origina11y published by Springer-Verlag Berlin Heidelberg New Vork in 1998.
The use of general descriptive names, registered names, trademarks, etc. in this pub
lication does not imply, even in the absence of a specific statement, that such names
are exempt from the relevant protective laws and regulations and therefore free for
general use.
Product liability: The publishers cannot guarantee the accuracy of any information
about the application of operative techniques and medications contained in this book.
In every individual case the user must check such information by consulting the rele
vant literature.
Data conversion: K+ V Fotosatz, Beerfelden
Cover Design: Mitterweger Medien GmbH, Plankstadt
SPIN 10687024. 18/3137-5 4 3 2 1 0 - Printed on acid-free paper
Trademarks
• Lumi-Imager™, LumiAnalyst™, TriPure™ are trademarks
of Boehringer Mannheim GmbH
• Windows ™ and Microsoft® Excel are a trademark and regis
tered trademark of Microsoft Corp.
• CSPD®, CDP-Star™, Western-Light™ and Nitro-Block™
are a registered trademark and trademarks of Tropix Inc.
• Stratalinker® and PosiBlot® are registered trademarks of
Stratagene Inc.
• Saran Wrap® is a registered trademark of Dow Chemicals
• ECL™ is a trademark of Amersham International PIe
• SYBR® green and SYPRO® red are registered trademarks of
Molecular Probes
• Protran® BA8S is a registered trademark of Schleicher and
Schuell
• PolyATract® is a registered trademark of Promega Corp.
I
Trademarks v
Digital imaging and quantification
of chemiluminescence and fluorescence:
Lumi-Imager and its applications *
Onno Bakker 1, Marti Aldea 2, Jörg Bergemann 3,
Albert Geiger4, Michael Kirchgesser5, Achim Kramer6,
• Stephan Schröder-Köhne 7, Hans-Joachim Höltke 8
ABSTRAa
The instrument, Lumi-Imager was developed for the detec
tion and accurate quantification of chemiluminescent signals
on blots and in microtiter plates. It contains a cooled CCD
camera, a specially developed lens system and software. In
addition to luminescence, Lumi-Imager can also detect and
quantify UV-excited fluorescent signals in gels. Here we de
scribe the components and features of this instrument, as
weil as so me of its applications.
* Parts of this article have been published in Onno Bakker et al., "Digital
imaging and quantification of chemiluminescence and fluorescence: detec
tion of mutation with protein truncation test". In: Practical use of non
radioisotopic systems. Part 11 - commercially available kits, ed. S. Nomura
and T. Watanabe (Tokyo: Shujunsha, 1998), 140-154
I Laboratory of Experimental Endocrinology, F5-171, Academic Medical
Centre, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
2 Dept. CiEmcies Mediques Basiques, Universitat de Lleida, Rovira Roure,
44, 25198 Lleida, Catalunya, Spain
3 Paul Gerson Unna Skin Research Center, Beiersdorf AG, Unnastraße 48,
20245 Hamburg, Germany
4 PNA Diagnostics AIS, R0nnegade 2, DK-2100 Copenhagen 0, Denmark
5 Boehringer Mannheim GmbH, Nonnenwald 2, 82372 Penzberg, Germany
6 Institut für Medizinische Immunologie, Universitätsklinikum Charite,
Schumannstraße 20-21, 10098 Berlin, Germany
7 Max-Planck Institut für biophysikalische Chemie, 37070 Göttingen,
Germany
8 Boehringer Mannheim GmbH, Nonnenwald 2, 82372 Penzberg, Germany
I
Digital imaging and quantification
AlM
The aim of the authors is to present the reader with their experience
in using Lumi-Imager, an apparatus recently developed by Boehringer
Mannheim to accurately quantify "glow-type" non-radioactive lumi
nescent signals.
INTRODUCTION
Non-radioactive methods are increasingly used in many laboratories
and are starting to replace traditional methods which involve radioac
tive isotopes for the labeling and detedion of nucleic acids and pro
teins. Non-radioadive methods not only avoid the hazards, cost, and
inconvenience associated with radioadive methods, but are also of
ten faster and more sensitive. Enzyme-catalyzed chemiluminescence
has established itself as the most sensitive non-radioactive detedion
method. Initially, chemiluminescence was widely used in Western
blotting applications. Non-radioactive haptens, such as digoxigenin
(DIG) or vitamin H (biotin), were later established for nucleic acid la
beling, whereby chemiluminescence proved to be highly sensitive in
applications such as Southern, Northern, dot blotting and gel-shift
assays. A disadvantage of these non-radioadive chemiluminescent
methods was that the signals had to be deteded with X-ray films.
Due to their very low dynamic ranges (approximately 1:100), these
films cannot simultaneously image very strong and very weak signals
without overexposure of the strong signal. Additionally, detecting sig
nals with X-ray films requires the use of a dark room, exposure to
be set up in a light-tight cassette, and films to be later developed.
This procedure entails extra work and inconvenience, and also in
volves the handling of hazardous chemicals. Furthermore, it is not
possible to directly quantitate signals on X-ray film. Indired quantita
tion is achieved by scanning the films, after wh ich absorbance values
can only then be taken as an indirect estimation of the intensity of
luminescence. As X-ray films exhibit a non-linear response to lumi
nescence (as weil as to radioactivity), data acquired in this mann er
can only provide a rough approximation of the true light emission
from the object. Nowadays, (CD cameras offer the possibility of sen
sitive detection and direct quantitation of light. In fact, deeply cooled
(CD cameras are used in astronomy to observe faint, distant objeds.
2 Digital imaging and quantification
Based on this technology, Boehringer Mannheim has developed
Lumi-Imager, a cooled CCD camera and special lens system for the
highly sensitive detection and accurate quantitation of chemilumines
cence on blots and in microtiter plates.
1.1 Technical description of the instrument
The system consists of a Peltier-cooled CCD camera (cooled to
about -40°C) and a specially developed lens system that allows
image acquisition of "glow-type" chemiluminescent signals,
e.g., catalyzed by alkaline phosphatase or horseradish peroxi
dase on sampies of up to 24x30 cm2 or four microtiter plates
(Fig. 1). The drawer can accommodates an UV-transillumina
tor, in which case a filter wheel with four different bandpass fil
ters is mounted in front of the lens system. The high resolution
detector (1280xl024 pixels) and specially designed optics
achieve aresolution of two lines per millimeter over the whole
Air Forced --=::::,: ::"""' __: --1
Cooling ii' i':
'.,!,J", GGO Chip with
Special . Tripie Stage Peltier
Lens ------1~~~~~ Cooling System
Filters: I ..
520 nm Mini Oarkroom
600 nm fI Enclosure
650 nm
Stabilized
..
UV trans
lIluminator -------1
Probe Size 20 cm x 20 cm
Fig. 1. Photograph and schematic drawing of the Lumi-Imager instrument
I
Digital imaging and quantification 3
sampIe area (24x30 cm2) and permit a fixed optical alignment.
This fixed optical alignment, together with a specially designed
focus stabilization which compensates for temperature varia
tions and long-term exposure effects, avoids tedious and re
peated focusing. These special optics and the built-in Win
dows-based camera driver software, which automatically per
forms flatfield- and detector-specific corrections, enable accu
rate quantitation of chemiluminescent signals across the entire
sampIe area. For microtiter plate measurements, Lumi-Imager
demonstrates excellent linearity over the whole dynamic range
and compares very favorably to dedicated luminescence microti
ter plate readers. With regards to contrast and resolution, sensi
tivity and image quality are wholly comparable to X-ray film.
In addition, Lumi-Imager provides the option to use a
more sensitive "binning" mode which reduces exposure time
bya factor of up to four compared to X-ray film; and although
resolution is reduced two-fold in the process, it is still compar
able to that of most phosphoimagers. The instrument has a dy
namic range of 1:10000 which allows for imaging and quantita
tive analysis of weak and strong signals in a single exposure.
To this end, LumiAnalyst software has been developed which
enables fast and accurate molecular weight determination and
quantification of bands and dots derived from all blotting ap
plications (Southern, Northern, dot and Western blots), as weIl
as microtiter plates, including calibration. Typical examples of
the LumiAnalyst screen are shown in Figs. 7 and 8. Experimen
tal data can be stored together with the images in an integrated
database. In addition, data can be transferred direct1y to
Microsoft® Excel for further analysis and generation of gra
phics. Furthermore, images are stored as TIFF files which can
be readily imported into other programs for documentation or
publication purposes, which, incidentaIly, was done with the
figures presented in this book.
I
1.2 Applications
Here we discuss some important cl,assic and widely used mo
lecular biology techniques in relatioit to non-radioactive label
ing and signal detection/quantification using Lumi-Imager. In
addition, more recent and exciting methods, such as the pro-
4 Digital imaging and quantifjcation