Discovery Matters
Issue 1 2005
Resolving truncated forms of histidine-tagged proteins
A demonstration of tandem affinity purification (TAP)
Automated protein data analysis for LC-MS/MS
siRNA screening of the cell cycle with dynamic GFP sensors
SPA beads for use with GST fusion substrates
Selective labeling of cell surface proteins with CyDye fluors
AND New products for your research
Discovery Matters
Issue 1 2005
Contents
3
What’s new?
Details of our newest products and services
8
Innovations forum
8
Scale-up and two-step purification of histidine-tagged proteins
with Ni Sepharose 6 Fast Flow
10
Tandem affinity purification (TAP) of two subcomplexes of a
transcription factor, TFIIH from Schizosaccharomyces pombe
Protein purification and production
14
A comparative study of Superdex 75 10/300 GL versus
TSK-GEL Super AW3000 for analytical gel filtration
16
Automated identification and quantitation of proteins and
peptides in mammalian samples using LC-MS/MS
18
siRNA screening of the cell cycle with two dynamic GFP sensors
20
SPA Imaging Charge-Based Binding Beads: A different approach
to the capture of assay product
Proteomics and protein analysis
Drug screening and cellular assays
12 Technology Central
High-throughput preclinical imaging
22 Technical Tips
22
Ettan DIGE: New protocol for selective labeling of cell surface
proteins using CyDye DIGE Fluor minimal dyes
23
Gravity-flow purification of histidine-tagged proteins using
Ni-Sepharose 6 Fast Flow packed in PD-10 columns
2 Discovery Matters Issue 1 2005 GE Healthcare
Welcome...
...to Discovery Matters from GE Healthcare. This
new publication replaces our former publication
Life Science News.
Why? Because we listened to you. Based on
feedback you provided, we’ve redesigned this
publication to make it easier to find the content
that’s most important to you. For example, we’ve
moved our new product announcement section
to the front of the magazine because you ranked
it as the most useful section. We’ve made the
articles more concise and provided them with an
easy-to-read layout and Web addresses that
take you directly to content online. After we
made these, and a few other changes, we
decided a new name was also in order.
Some things are still the same. The "What’s
New?" section delivers brief announcements of
our latest product releases. "Innovations Forum"
is where you will find articles about the
development, application, and use of new
products or interesting uses of existing products.
"Technology Central", found in the center of the
magazine, provides more graphical and technical
information about a product or related groups of
products. The "Technical Tips" section provides
summaries of a new or modified use of a product
and protocol enhancements.
The goal of Discovery Matters is to provide you
with information that will help you achieve your
research objectives. We want to continue
developing Discovery Matters into a publication
you value, and we’d very much like to continue
receiving your input. Please send any comments,
questions, or concerns, as well as submissions
of articles you’d like to publish to
[email protected].
what’s new?
shop online www.amershambiosciences.com
HisTrap FF crude columns
• Reliable purification of histidine-tagged proteins
directly from unclarified lysates—simply sonicate and
run your sample.
• Reduces sample preparation time, which minimizes
protein degradation.
• Prepacked with Ni Sepharose™ 6 Fast Flow, which has high
protein binding capacity and negligible Ni2+ ion leakage.
• Compatible with a wide range of reducing agents,
detergents, denaturants, and other additives.
• Available in convenient prepacked 1-ml and 5-ml HiTrap™
column formats.
• Simple operation with a syringe, pump, or chromatographic system such as
ÄKTAdesign™
or
FPLC™
System.
HisTrap™ FF crude columns offer one step purification of
histidine-tagged proteins from unclarified lysates. The specific
design of the columns eliminates the need for centrifugation
and filtration of samples before loading.
For more information, visit
www.amershambiosciences.com/his
HisTrap FF crude (5 × 1 ml)
11-0004-58
HisTrap FF crude (100 × 1 ml)*
11-0004-59
HisTrap FF crude (5 × 5 ml)
17-5286-01
HisTrap FF crude (100 × 5 ml)*
17-5286-02
* Pack size available by special order.
Tricorn Coarse Filter Kit
Tricorn™ Coarse Filter Kit is an accessory designed to improve
the performance of Tricorn columns in laboratory-scale
applications. The filter included in the kit is made of robust,
high-density polyethylene, which is designed to reduce clogging
in the purification of large sample volumes or repeated loading
of clarified feed. The filter kit is particularly suited for use with
Tricorn columns packed with capture media based on matrices
such as Capto or Sepharose Fast Flow.
Tricorn Coarse Filter Kit includes five top and bottom filters, five
EPDM O-rings, and instructions.
For more information on Tricorn columns and accessories,
visit www.amershambiosciences.com/tricorn
Tricorn 5 Coarse Filter Kit
11-0012-53
Tricorn 10 Coarse Filter Kit
11-0012-54
Discovery Matters Issue 1 2005 GE Healthcare 3
what’s new?
Capto Q—raising productivity at high flow
• High-throughput, strong anion exchange medium for
capture and intermediate purification at process scale.
• Combined high binding capacity, high flow rate, and low
backpressure properties reduce process cycle times and
improve productivity.
• Rigid agarose matrix allows high bed heights and
purification of viscous samples at high flow rates.
shop online www.amershambiosciences.com
increased productivity. As a BioProcess™ medium, Capto Q
meets the demands of biopharmaceutical manufacturers for
fast, efficient, and cost-effective protein purification. Capto Q is
also available in HiTrap columns to save you time and sample
during process development. The HiTrap format allows you to
screen suitable media and develop basic separation methods.
To request a data file, please visit
www.amershambiosciences.com/captoq
• Cost-effective processing with smaller unit operations.
Capto™ Q is a strong anion exchange medium that increases
speed and throughput in capture and intermediate purification.
It combines high capacity with high flow velocity and low
backpressure, resulting in reduced process cycle times and
Updated Biotrak Web site
• Order online to save time and effort.
• Select assays either by using the new disease state
selection guide or by analyte type.
• Search for product information that is relevant to
your research.
The updated Biotrak Web site is a valuable source of
information on our range of Biotrak assays. It is now possible
to search either for assays that support the research of a
particular disease state or by analyte type. A list of published
articles is also available if you need more details on how
Biotrak assays can support your application.
To see how these updates can help you, visit
www.amershambiosciences.com/biotrak
4 Discovery Matters Issue 1 2005 GE Healthcare
Capto Q (25 ml)
17-5316-10
Capto Q (500 ml)
17-5316-01
HiTrap Capto Q (5 × 1 ml)
11-0013-02
HiTrap Capto Q (5 × 5 ml)
11-0013-03
what’s new?
shop online www.amershambiosciences.com
on bench time is reduced by at least 60 min compared with
Medsystem’s standard format ELISA.
Biotrak Easy ELISAs
• Simplified format: All the main assay components
are supplied lyophilized and preloaded on a coated
96-well microplate*.
• Fewer steps: Faster and easier to perform than standard
Assays are performed through the simple addition of distilled
water to standard wells, or diluted samples to the sample wells.
Following the addition of TMB substrate, the reaction is stopped
and the results are read at 450 nm on a microplate reader.
format ELISAs.
* Each Easy ELISA microplate is supplied with coating antibody and preloaded with the
• Equivalent performance: Innovative assay design delivers
results that are equivalent to standard format ELISAs.
Easy ELISA technology enables the simple and rapid
quantitation of antigen in a range of sample matrices. Because
the assay reagents are supplied ready to use, there is no need
for standard curve titration or pre-dilution of reagents. Hands-
following lyophilized components: detection antibody, streptavidin-HRP (if necessary),
and sample diluent.
For more information, visit
www.amershambiosciences.com/biotrak and click
“Assay Categories”
Easy ELISA format
IFNα Human, Biotrak Easy ELISA (96 wells)
Dehydrated
components
IFNγ Human, Biotrak Easy ELISA (96 wells)
RPN5961
IL-1β Human, Biotrak Easy ELISA (96 wells)
RPN5971
IL-2 Human, Biotrak Easy ELISA (96 wells)
RPN5965
IL-2 Mouse, Biotrak Easy ELISA (96 wells)
RPN5966
IL-6 Human, Biotrak Easy ELISA (96 wells)
RPN5968
IL-8/NAP-1 Human, Biotrak Easy ELISA (96 wells)
RPN5969
IL-10 Human, Biotrak Easy ELISA (96 wells)
RPN5962
IL-10 Mouse, Biotrak Easy ELISA (96 wells)
RPN5963
MCP-1 Human, Biotrak Easy ELISA (96 wells)
RPN5964
IL-13 Human, Biotrak Easy ELISA (96 wells)
RPN5972
TGFβ1 Human, Biotrak Easy ELISA (96 wells)
RPN5970
TNFα Human, Biotrak Easy ELISA (96 wells)
RPN5967
TNFβ Human, Biotrak Easy ELISA (96 wells)
RPN5973
Rehydration
Add sample
Incubation and
washing
Standard ELISA format
Coated
microwell
+
+
Second
incubation
and
washing
First
incubation
and
washing
monoclonal coating antibody
antigen
biotin conjugate
streptavidin-HRP
reacted substrate
RPN5960
Discovery Matters Issue 1 2005 GE Healthcare 5
what’s new?
shop online www.amershambiosciences.com
HitHunter Enzyme Fragment
Complementation Assays
• Single-label technology based on enzyme fragment
complementation (EFC).
• Separate assays for chemiluminescence and red-shifted
fluorescence detection.
• Require no mixing steps and limited number of
HitHunter™ Enzyme Fragment Complementation (EFC) Assays
are competitive binding assays based on the recombination
of two inactive enzyme fragments to form the active
tetrameric β-galactosidase. HitHunter EFC Assays are
amenable to automation and suitable for high-throughput
screening applications.
reagent additions.
Visit www.amershambiosciences.com/promo_dm012
for more information
cAMP HitHunter Fluorescence Assay for Adherent Cells1, 2
Estrogen Receptor HitHunter Fluorescence Assay1
100 assays; 96 well
90-0001-01
100 assays; 96 well
90-0020-01
800 assays; 384 well
90-0001-02
800 assays; 384 well
90-0020-02
cAMP HitHunter Chemiluminescence Assay for Adherent Cells1, 2
Estrogen Receptor HitHunter Chemiluminescence Assay1
100 assays; 96 well
90-0003-01
100 assays; 96 well
90-0019-01
800 assays; 384 well
90-0003-02
800 assays; 384 well
90-0019-02
cAMP HitHunter Fluorescence Assay for Cells in Suspension1, 2
Progesterone Receptor HitHunter Fluorescence Assay1
100 assays; 96 well
90-0002-01
100 assays; 96 well
800 assays; 384 well
90-0002-02
800 assays; 384 well
cAMP HitHunter Chemiluminescence Assay for Cells in
Suspension1, 2
Progesterone Receptor HitHunter Chemiluminescence
90-0018-01
90-0018-02
Assay1
100 assays; 96 well
90-0004-01
100 assays; 96 well
90-0017-01
800 assays; 384 well
90-0004-02
800 assays; 384 well
90-0017-02
cAMP II HitHunter Chemiluminescence Assay1, 3
Serine/Threonine Kinase HitHunter Fluorescence Assay1
100 assays; 96 well
90-0034-01
100 assays; 96 well
800 assays; 384 well
90-0034-02
1200 assays; 384 well
10 000 assays; 1536 well
90-0032-03
50 000 assays; 1536 well
90-0032-05
cAMP XS HitHunter Chemiluminescence
Assay1, 4
100 assays; 96 well
90-0041-02
Assay1
100 assays; 96 well
90-0027-01
1200 assays; 384 well
90-0027-02
Caspase 3 HitHunter Chemiluminescence Assay1
100 assays; 96 well
90-0028-01
1200 assays; 384 well
90-0028-02
cGMP HitHunter Chemiluminescence Assay1
100 assays; 96 well
800 assays; 384 well
90-0015-01
1200 assays; 384 well
90-0015-02
Tyrosine Kinase HitHunter Fluorescence Assay1
90-0010-01
1200 assays; 384 well
90-0010-02
Tyrosine Kinase HitHunter Chemiluminescence Assay1
100 assays; 96 well
90-0011-01
1200 assays; 384 well
90-0011-02
1
10 000 and 40 000 assays; 384-well pack sizes also available. See
www.amershambiosciences.com for ordering information.
2
A reliable and simple method for the detection of cAMP in cells.
3
Features a rapid two-step protocol for monitoring cellular activation of G proteincoupled receptors (GPCRs), particularly Gs coupled receptors.
4
Features high signal-to-background ratios for monitoring cellular activation of GPCRs,
particularly Gi coupled GPCRs
Cortisol HitHunter Chemiluminescence Assay1
100 assays; 96 well
90-0040-01
800 assays; 384 well
90-0040-02
6 Discovery Matters Issue 1 2005 GE Healthcare
100 assays; 96 well
90-0039-01
90-0039-02
90-0013-02
Assay1
100 assays; 96 well
90-0041-01
800 assays; 384 well
Caspase 3 HitHunter Fluorescence
Serine/Threonine Kinase HitHunter Chemiluminescence
90-0013-01
what’s new?
shop online www.amershambiosciences.com
DeCyder Extended Data Analysis (EDA) Software
• Powerful new analysis tool, extends the statistical options
offered in 2-D DIGE (2-D difference gel electrophoresis).
• Switches between statistical results and visualization
data from DeCyder™ 2-D Differential Analysis Software
seamlessly.
• Presents multivariate analysis of Ettan™ DIGE results, for
example using hierarchical cluster analysis, K-means
analysis, and principal component analysis (PCA).
• Links to internal and external databases, enabling results
to be put in biological context.
DeCyder EDA Software offers advanced statistical analysis in a
simple-to-use format, uncovering patterns in expression data
and relationships using multivariate analysis and sophisticated
clustering methods. The software aids understanding of
regulatory pathways, finds proteins with similar expression
profiles, or groups samples according to common expression
patterns. DeCyder EDA can also identify proteins that are
expressed differentially among disease states, tumor types, or
other sample subtypes.
DeCyder EDA Software
11-0026-82
DeCyder MS Differential Analysis Software
• Provides a new level of software support for MS-based
differential analysis, without the need for mass tag
chemistry.
• Visualizes LC-MS data in a novel way for effective
navigation and presentation of large datasets.
• Reduces hands-on time and minimizes user-to-user
variation.
• Automatically presents quantitative information and
matches statistics from complex experiments such as
time-dose series.
• Integrates with common protein identification
search engines.
DeCyder MS Differential Analysis Software significantly
improves efficiency through automatic detection, matching, and
analysis of peptides from multiple LC-MS experiments. The
software detects small quantitative differences between
peptides with high statistical confidence.
Visit www.amershambiosciences.com/promo_dm004
to download an application note
DeCyder MS Differential Analysis Software
11-0013-32
(including PC and single concurrent network user license)
DeCyder MS Differential Analysis Software
11-0013-31
(including single concurrent network user license)
Discovery Matters Issue 1 2005 GE Healthcare 7
Innovations Forum: Protein purification and production
Scale-up and two-step purification of histidine-tagged
proteins with Ni Sepharose 6 Fast Flow
J. Lundqvist, K. Torstenson, and L. C. Andersson
GE Healthcare, Uppsala, Sweden
Scaling up the purification of (histidine)6-tagged maltose
binding protein (Mr 43 000) with prepacked HisTrap™ FF and
HisPrep™ 16/10 FF columns (1-ml to 20-ml column formats)
was achieved without change in purity or loss of recovery.
Moreover, a two-step purification of N-terminal (histidine)10tagged Trx-P450 (Mr 133 200) using a HisTrap FF column
for the first step and gel filtration for the second step is
described. With this approach, the full-length target protein
could be separated from two histidine-tagged truncated
forms of the target protein.
Introduction
Immobilized metal ion affinity chromatography (IMAC) exploits
the interaction between chelated transition metal ions and sidechains of certain amino acids (mainly histidine) on proteins✧.
In general, nickel (Ni2+) is the preferred and most commonly
used metal ion for purification of histidine-tagged proteins.
The new IMAC medium, Ni Sepharose 6 Fast Flow, consists of
90-µm beads of highly cross-linked agarose, to which a
chelating group has been coupled and charged with Ni2+ ions.
The medium provides high binding capacity of histidine-tagged
proteins while maintaining low leakage of Ni2+.
In this article, scale-up and two-step purifications of histidinetagged proteins using Ni Sepharose 6 Fast Flow are described.
Scale-up purification
Figure 1 shows scale-up purification from HisTrap FF 1-ml via
HisTrap FF 5-ml to HisPrep FF 16/10 (20-ml) prepacked columns.
The sample used was an E. coli extract containing (histidine)6tagged maltose binding protein (MBP-[His]6, Mr 43 000). Pooled
fractions analyzed by SDS-PAGE showed almost identical purity
for each of the three columns and the recovery did not change.
Scale-up purification can also be achieved by coupling
HisTrap FF 1-ml or 5-ml columns in series.
A two-step purification of (Histidine)10-tagged Trx-P450
(Histidine)10-tagged Trx-P450 ([His]10-Trx-P450) is an N-terminal
histidine-tagged fusion of thioredoxin 1 (from E. coli) and
cytochrome P450 reductase (from Bacillus megaterium, total
8 Discovery Matters Issue 1 2005 GE Healthcare
Mr ≈133 200, 1192 aa). This protein was purified using a twostep protocol (Fig 2): IMAC using HisTrap FF 1 ml was employed
for the first step; and gel filtration with HiLoad™ 16/60
Superdex™ 200 prep grade was used in the second step.
The IMAC purification was performed by applying 50 ml
of E. coli extract onto the column (Fig 2A); 5.2 ml of eluted
sample was applied to the gel filtration column for the
second step of the purification (Fig 2B).
Gel filtration of the IMAC pool (Fig 2B) gave three peaks,
representing (His)10-Trx-P450 and two contaminants (peaks 1–3
respectively). The two contaminants co-purified with the target
protein by IMAC were shown by N-terminal Edman sequencing
to be truncated forms of the target protein, each with an intact
N-terminal (histidine)10 tag. The truncated forms were easily
separated from the full-length target protein by gel filtration as
confirmed by SDS-PAGE (Fig 2C).
Conclusion
Scaling up column dimensions from HisTrap FF 1 ml via HisTrap
5 ml to HisPrep FF 16/10 did not affect purity or recovery.
(His)10-Trx-P450 was purified using a two-step protocol; IMAC
followed by gel filtration. It was possible to completely separate
two truncated forms of the target protein from the full-length
target protein.
To obtain a data file on these products, please visit
www.amershambiosciences.com/promo_dm001
Ordering Information
HisTrap FF 1 ml (5 × 1 ml)*
17-5319-01
HisTrap FF 5 ml (5 × 5 ml)*
17-5255-01
HisPrep FF 16/10 (1 × 20 ml)
17-5256-01
Ni Sepharose 6 Fast Flow (25 ml)
17-5318-01
Ni Sepharose 6 Fast Flow (100 ml)*
17-5318-02
* Larger pack sizes are available.
To shop online, go to www.amershambiosciences.com
✧
See licensing information on page 24.
Innovations Forum: Protein purification and production
A
Columns:
Sample:
as indicated
Histidine-tagged maltose binding
protein in E. coli extract
Binding buffer: 20 mM sodium phosphate, 25 mM
imidazole, 500 mM NaCl, pH 7.4
Elution buffer: binding buffer + 500 mM imidazole
Flow rates:
HisTrap FF 1 ml, 1 ml/min;
HisTrap FF 5 ml, 5 ml/min;
HisPrep FF 16/10, 5 ml/min
System:
ÄKTAexplorer™ 100
mAU
HisTrap FF, 1 ml
4000
6.2 mg
3000
2000
Fig 1. Scale-up from
HisTrap FF 1 ml via
HisTrap FF 5 ml to a
HisPrep FF 16/10 (20 ml)
prepacked column.
(A) The samples loaded
contained approximately
8, 40, and 160 mg MBP(His)6, respectively.
Recovery in milligram
is shown in each
chromatogram.
(B) SDS-PAGE (ExcelGel™
SDS Gradient 8–18)
confirms that scaling
up from the 1-ml to the
20-ml column did not
affect the purification
result. M = low molecular
weight marker.
A
Column:
Sample:
HisTrap FF 1ml
50 ml E. coli extract with
(His)10-Trx-P450
Binding buffer: 20 mM sodium phosphate,
500 mM NaCl, 60 mM
imidazole, pH 7.4
Elution buffer: binding buffer + 500 mM imidazole
Flow rate:
1–1.5 ml/min
System:
ÄKTAexplorer 100
mAU
4000
3000
2000
1000
0
1000
0.0
20.0
40.0
60.0
80.0
ml
0
0.0
5.0
10.0 15.0 20.0 25.0 30.0
ml
B
Column:
Sample:
HiLoad 16/60 Superdex 200 prep grade
5.2 ml sample from IMAC purification
shown in (A)
Eluent:
20 mM sodium phosphate, 280 mM
NaCl, 6 mM KCl, pH 7.3
Flow rate: 1 ml/min
System:
ÄKTAexplorer 100
HisTrap FF, 5 ml
mAU
4000
33 mg
3000
2000
mAU
Fig 2. Two-step
purification of (His)10-TrxP450 from E. coli extract.
(A) First-step IMAC using
HisTrap FF 1 ml. The
buffer used for
equilibration, sample
application, and wash
contained 60 mM
imidazole. The arrows
mark the peak that was
collected and pooled.
(B) The pool eluted in the
IMAC step was applied
on a HiLoad 16/60
Superdex 200 prep grade
column. Peak 1 = full
length protein, peak 2 =
truncated protein, peak
3 = truncated protein.
(C) SDS-PAGE (ExcelGel
SDS Gradient 8–18)
shows (His)10-Trx-P450
and two truncated forms
of this protein (corresponding to peaks 1, 2
and 3 respectively from
the gel filtration
purification). M = low
molecular weight
marker. Ratios indicate
dilution factor of sample
loaded onto gel.
1000
0
0.0
50
100
150
ml
HisPrep FF 16/10, 20 ml
mAU
4000
3000
4000
149 mg
3000
2000
2000
1000
3
1
2
1000
0
0
0
100 200 300 400 500 600
ml
20.0
B
M
HisTrap HisPrep
Start
material 1 ml 5 ml 16/10
40.0
C
M
Mr
(× 10-3)
97
66
60.0
80.0
100.0
ml
IMAC
Gel filtration
IMAC Peak Peak Peak
Flow2
3
1
Start through Wash pool
(1:2)
(1:20) (1:20) (1:10) (1:10) (1:2) (1:2)
M
Mr
(× 10-3)
97
66
45
45
30
20.1
30
20.1
14.4
14.4
Discovery Matters Issue 1 2005 GE Healthcare 9
Innovations Forum: Protein purification and production
Tandem affinity purification (TAP) of two
subcomplexes of a transcription factor,
TFIIH from Schizosaccharomyces pombe
H. Spåhr* and R. Bhikhabhai†
* Karolinska Institute, Stockholm, Sweden
† GE Healthcare, Uppsala, Sweden
Two subcomplexes of TFIIH, core-TFIIH and TFIIK, in
Schizosaccharomyces pombe were purified with the tandem
affinity purification (TAP) method. A modified version of the
TAP protocol originally developed by Seraphin and coworkers
was used. The purification was performed in high- and lowstringency conditions using 1 ml each of the affinity media
IgG Sepharose™ 6 Fast Flow and Calmodulin Sepharose 4B.
The individual subunits of the subcomplexes were identified
with MALDI-ToF mass fingerprinting and the activity was
measured in vitro by kinase and transcription assays.
Associated
proteins
Contaminants
C
Experimental procedure
Pmh1-TAP and spTfb2-TAP
The TAP tag was attached at the C-terminus of Pmh1 to purify
TFIIK and at the C-terminus of spTfb2 to purify core-TFIIH.
A summary of the sample preparation and subcomplex
purification is described in detail by Spåhr et al (1). Information is
also available on the TAP method Web site (4).
Modifications to the TAP protocol
For both steps, the affinity matrix was incubated with the
sample and then packed into a plastic column.
10 Discovery Matters Issue 1 2005 GE Healthcare
Prot A
C
TEV Protease
cleavage site
Target
protein
C
C
Binding
C
First affinity
column
IgG beads
Introduction
For yeast and mammalian cells, the minimum set of general
transcription factors needed for in vitro basal transcription,
include RNA polymerase II, TATA-binding protein and transcription factors (TFs) IIB, IIE, IIF, and IIH. This report describes the
purification of two transcription factor subcomplexes from the
yeast Schizosaccharomyces pombe; core-TFIIH and TFIIK of
transcription factor IIH (1). The complexes were purified by using
a modified version of the tandem affinity purification (TAP)
protocol originally developed by Seraphin and coworkers
(2, 3; Fig 1). To recover all the proteins of core-TFIIH and TFIIK,
a TAP-tag was introduced at the carboxyl terminus of subunits
spTfb2 and Pmh1 respectively.
C Calmodulin binding peptide
C
Fig 1. Overview of the
Tandem Affinity
Purification (TAP)
strategy. The TAP tag
comprises CBP
(Calmodulin binding
peptide) linked to a
Protein A domain
separated by a TEV
cleavage site. Permission
to reprint this figure was
granted by Elsevier,
Global Rights
Department.
TEV protease
cleavage
C
C
Second affinity
column (Ca2+)
Calmodulin
beads
Native elution
EGTA
High stringency conditions
Purification of core-TFIIH and TFIIK was made in high stringency
conditions. The original protocol was modified so that 1 ml,
instead of 200 µl, of affinity media was used. For core-TFIIH, the
sodium chloride concentration was increased from 150 mM to
500 mM in the elution buffer.
Low stringency conditions
A low stringency protocol was used to co-purify additional
proteins that interacted weakly with TFIIK (Pmh1-TAP). The
concentration of detergent, Nonidet P-40 was decreased from
0.1% to 0.01% throughout the purification procedure. Moreover,
the buffer volumes used to wash the IgG Sepharose and
Calmodulin Sepharose (GE Healthcare) were decreased
from 30 to 5 column volumes.
Innovations Forum: Protein purification and production
250
250
250
B
150
150
100
100
C
D
150
Rad 15
75
IK
Ho
lo
-T
FI
100
TF
I
Ercc3sp
75
co
nt
r
ol
IH
A
75
spTfb1
Pmh1
50
50
}
Mcs6
Mcs2
37
25
Ef1a
Pmh1
Actin
spTfb2
spSsl1
37
}
Mcs6
Mcs2
50
CTD
37
spTfb4
*
25
25
Fig 2. Characterization of TFIIH. Proteins were separated on a 10% SDS-PAGE gel,
visualized by Coomassie Brilliant Blue, and identified with MALDI-TOF mass fingerprinting. Arrows on the left side of each lane indicate identified proteins, and arrows on
the right indicate molecular masses according to standard. (A) Three subunits of TFIIK
(Pmh1-TAP) when purified using the high stringency conditions. (B) Rad15 associated
with TFIIK (Pmh1-TAP) when low stringency conditions were used. The major
contaminants Actin and Ef1a are indicated in the figure. (C) core-TFIIH (spTfb2-TAP)
was purified using high stringency protocol. The band below spTfb4 (*) is a contaminant. (D) TFIIK phosphorylates the C-terminal domain (CTD) of RNA polymerase II. The
figure is published with kind permission of American Society of Biochemistry and
Molecular Biology.
Results and Discussion
S. pombe alcohol dehydrogenase promoter (1). In addition,
TFIIK could phosphorylate the C-terminal domain (CTD) of
RNA polymerase II. Core-TFIIH did not notably influence
the CTD kinase activity (Fig 2D).
TFIIK
The initial aim was to purify transcription factor IIH from
S. pombe as a complex of nine subunits as earlier identified
in S. cerevisiae. Under high stringency conditions, the results
showed that only three subunits (Fig 2A) co-purified instead of
the expected nine. The individual subunits were identified with
MALDI-ToF mass fingerprinting as Mcs2, Mcs6, and Pmh1. As a
similar trimeric complex is found in S. cerevisiae, this complex
was denoted spTFIIK.
In a second attempt to purify all nine subunits of TFIIH, a
protocol with low stringency conditions, with a lower detergent
concentration was used. When analyzed, substoichiometric
quantities of Rad15 (a homologue to the Rad3 helicase from
S. cerevisiae) together with three subunits of TFIIK and two
major contaminants, actin and Ef1a were identified (Fig 2B).
No other components of core-TFIIH were found. This indicates
that TFIIH may be a less stable complex in S. pombe than has
been described in S. cerevisiae and human cells.
core-TFIIH
To purify core-TFIIH, the high stringency protocol was
employed. Core-TFIIH was shown to contain five subunits,
Ercc3sp, spTfb1, spTfb2, spSsl1 and spTfb4 (Fig 2C). The sixth
protein, Rad15 was not co-purified under these conditions.
Activity of TFIIH subcomplexes
The presence of TFIIK and core-TFIIH increased basal transcription levels on a supercoiled template under the control of the
Conclusions
The tandem affinity purification method allows purification
of native protein complexes using mild binding and elution
conditions. The amounts of purified TFIIH subcomplexes in
S. pombe was sufficient for subunit identification by MALDI-ToF
mass fingerprinting and for use in in vitro activity assays.
References
1. Spåhr, H. et al. Mediator Influences Schizosaccharomyces pombe RNA Polymerase IIdependent Transcription in vitro. J. Biol. Chem. 278, 51301–51306 (2003).
2. Rigaut, G. et al. A generic protein purification method for protein complex
characterization and proteome exploration. Nature Biotech. 17, 1030–1032 (1999).
3. Puig, O. et al. The tandem affinity purification (TAP) method: a general procedure of
protein complex purification, Methods 24, 218–29 (2001).
4. TAP method home page: http://www-db.embl-heidelberg.de/jss/servlet/
de.embl.bk.wwwTools.GroupLeftEMBL/ExternalInfo/seraphin/TAP.html
To view this article online, go to
www.amershambiosciences.com/promo_dm002
Ordering Information
Calmodulin Sepharose 4B (10 ml)
17-0529-01
IgG Sepharose 6 Fast Flow (10 ml)
17-0969-01
To shop online, go to www.amershambiosciences.com
Discovery Matters Issue 1 2005 GE Healthcare 11
technology central
High-throughput preclinical imaging
Optimized workflow and data management
Short scan times simplify animal handling by reducing
requirements for extended anesthesia, temperature
regulation, respiratory gating or ventilation, and animal care
technician time. Additionally, images from the eXplore Locus
Ultra export seamlessly into clinical workstations, such as
our Advantage Workstation™, to further streamline data
management and workflow.
Integrated platform for diverse applications
The eXplore Locus Ultra provides an integrated software
and hardware solution to address the most challenging
research applications.
Perfusion
Tumor: Measure parameters such as blood flow, blood volume,
mean transit time, and tissue permeability. Correlate blood flow
dynamics to tumor growth and necrosis.
The eXplore Locus Ultra is a high-throughput preclinical
CT scanner enabling anatomical and physiological in vivo
small animal studies. A combination of flat-panel detector
technology and a clinical class X-ray tube ensures
unmatched acquisition speed and image quality (1).
Inflammation: Quantitate changes in tissue flow or volume
resulting from immune response activation related to injury or
disease onset.
Organ: Track changes in function and viability of organs such as
the kidney, heart, or lung.
High performance
One-second volume data acquisition permits use of
short-lived common contrast agents and provides highthroughput imaging. In addition, a large transaxial field of
view (14 cm) and long axial coverage (up to 10 cm/rotation)
accommodate diverse animal models.
Gray scale
Mean transfer time
Blood flow
Blood volume
Consistent and accurate analysis
The eXplore Locus Ultra provides high-quality images with
contrast-to-noise, resolution, and dose performance optimized
for preclinical imaging.
Multimodality imaging
The eXlore Locus Ultra shares a collection of compatible animal
beds with eXplore Vista (preclinical PET) and eXplore Optix
(preclinical optical) for accurate coregistration of superimposed
images. With this combination of functional and anatomical
information, biochemical activities can be detected, quantitated,
monitored, and registered to a specific location.
12 Discovery Matters Issue 1 2005 GE Healthcare
Rat brain perfusion.
Spectrum colors from
blue to red denote lower
to higher parameters
respectively.
Acknowledgements:
Ting Y Lee, Jennifer
Hadway, L. Du, S. van
Doodewaard, P. Picot
and W. Ross, Robarts
Research Institute,
Lawson Health Research
Institute, University of
Western Ontario, and
GE Healthcare.
Oncology
Angiogenesis: Observe hyper-vascularization and vessel
morphology that differentiates normal vessels from tumorfeeding vessels to quantitate disease progression (2). The
eXplore Locus Ultra can evaluate the efficacy of antiangiogenic therapies in longitudinal in vivo studies.
Differentiate normal
tissues from tumors
through analysis of
vascularization and
permeability parameters.
Acknowledgements:
S. Greschus, Department
of Neuroradiology,
University Giessen;
F. Kiessling, German
Cancer Research
Center, Heidelberg.
Respiratory disease
Visualize and quantitate airway structures in small animals.
The short acquisition time dramatically reduces image
artifacts due to lung or abdominal motion, without the need
for gating. This is especially convenient in following tumor
metastases longitudinally thereby reducing animal usage
and operating cost.
Bone disease
Osteoporosis: Rapidly assess disease development,
progression, and therapeutic efficacy with true volume
bone mineral density measurements.
Arthritis: Monitor anatomical changes indicative of disease
progression (e.g., joint spacing, density changes in bone and
subchondral bone).
Cardiovascular disease
Apply common contrast agents to study stenosis, vascular
disease and development, vascular injury and repair, vessel
geometry, and therapeutic efficacy. Blood flow and blood
volume measurements could potentially be related in
estimating the extent of ischemia and tissue viability.
Gray scale
Blood flow
Mean transfer time
Blood volume
Slice (0.45 mm) from a
normal mouse heart. The
grayscale image is the
average of CT images of
the same slice acquired
during the first passage
of contrast media.
Acknowledgements:
Ting Y Lee, Jennifer
Hadway, L. Du, S. van
Doodewaard, P. Picot
and W. Ross, Robarts
Research Institute,
Lawson Health Research
Institute, University of
Western Ontario.
Whole body imaging of a neonatal mouse. High resolution, volume rendering of a
mouse skeleton without using contrast agents.
Phenotyping
Characterize anatomical differences in transgenic models and
quantitate the resulting changes in longitudinal studies. Imaging
the same live animal at multiple time points increases research
productivity and reduces the number of animals required.
References
1. Kiessling, F. et al. Volumetric computed tomography (VCT): a new technology for
noninvasive, high-resolution monitoring of tumor angiogenesis. Nature Med. 10,
1133–1138 (2004).
2. Lee, T. Y. et al. CT imaging of angiogenesis. Q. J. Nucl. Med. 47,171–187 (2003).
For a brochure on the eXplore Locus Ultra, please visit
www.amershambiosciences.com/promo_dm008
Discovery Matters Issue 1 2005 GE Healthcare 13
Innovations Forum: Protein purification and production
A comparative study of Superdex 75 10/300 GL versus
TSK-GEL Super AW3000 for analytical gel filtration
F. Calais and H. Hedlund
GE Healthcare, Uppsala, Sweden
Superdex™ 75 10/300 GL and TSK-GEL™ Super AW3000 are
prepacked columns for analytical gel filtration. The two
nevertheless differ considerably in several key aspects:
column length, matrix material, particle size and porosity, for
example. This report compares their performance to see how
these differences affect resolution and analysis time for
fractionating a standard protein mixture.
Introduction
A number of prepacked columns for analytical gel filtration
are available. One example is GE Healthcare’s Superdex 75
10/300 GL, which comprises Superdex 75 packed in a 30 cm
Tricorn™ High Performance 10/300 GL column. Superdex 75
is a composite matrix of dextran and agarose with a porous
structure and an average particle size of 13 µm. It gives high
resolution for proteins and peptides in the molecular weight
range 3000 to 70 000.
Tosoh Bioscience has recently launched a new gel filtration
column series for analytical work. Called the TSK-GEL Super AW
series, these columns feature a 15 cm bed height and a
polymer-based matrix with a 4 µm particle size. Because of
these two features, the TSK-GEL Super AW3000 column
(fractionation range up to 60 000) is said to give the resolution
equivalent to a 30 cm column like Superdex 75 10/300 GL yet in
approximately half the time.
The protein test sample mixture specified in Table 2 was filtered
through a 0.45 µm filter and the columns conditioned for initial
use with 2 column volumes (CV) of buffer (0.05 M sodium
phosphate, 0.15 M NaCl, pH 7.0). Following column equilibration
with 0.2 CV of buffer, the protein test mixtures were injected
onto the respective columns and then eluted by isocratic elution
within 1.5 CV. Both columns were run at the same linear flow
rate. Table 3 lists more details. Figures 1 A and B show the
resulting chromatograms for Superdex 75 10/300 GL and
TSK-GEL Super AW3000 respectively.
Table 1. Characteristics of Superdex 75 10/300 GL and TSK-GEL Super AW3000 columns.
Superdex 75 10/300 GL
Particle size
TSK-GEL Super AW3000
13–15 µm
4 µm
Separation range
3000–70 000
Up to 60 000
No. of theoretical plates
≥ 30 000 m-1
≥ 16 000 m-1
10 × 300
6 × 150
i.d. × bed height (mm)
Bed volume (ml)
Max. operating back pressure
Max. flow (ml/min)
Column material
24
4.2
1.8 MPa
6.0 MPa
1.5
0.6
Glass
Steel
Table 2. Test sample mixture.
MW
In this study, the two columns were compared with regard to
analysis time and resolution when separating a model test
sample mixture.
Separation of protein test sample
mixture using ÄKTApurifier
Table 1 summarizes the main characteristics of the two
columns. Table 2 lists the components mixed as a test sample to
compare the columns.
The chromatographic system used was ÄKTApurifier™ equipped
with a UV flow cell (10 mm pathlength) and 0.6 ml mixer. UV
detection was at 280 nm. The system was controlled using
UNICORN™ control software.
14 Discovery Matters Issue 1 2005 GE Healthcare
Concentration
BSA*
67 000
8 mg/ml
Ovalbumin
43 000
2.5 mg/ml
Ribonuclease A
13 700
5.0 mg/ml
Aprotinin
6512
0.5 mg/ml
Vitamin B12
1355
0.1 mg/ml
* Note that BSA will elute in the void volume of TSK-GEL Super AW3000.
Results
Superdex 75 10/300 GL column had the highest resolution, while
the shorter TSK-GEL Super AW 3000 column had the fastest run
time (Fig 1; TSK-GEL column 1 CV = 14 min compared with
1 CV = 28 min for Superdex 75 10/300 GL). In addition, TSK-GEL
Innovations Forum: Protein purification and production
Column:
Sample:
Eluent:
Flow rate:
System:
Factors influencing resolution in gel filtration
Superdex 75 10/300 GL
50 µl (0.21% CV) protein mixture (Table 2)
50 mM sodium phosphate, 150 mM NaCl, pH 7.0
0.85 ml/min (65 cm/h)
ÄKTApurifier
BSA
mAU
mS/cm
ovalbumin
28.0
ribonuclease A
150
aprotinin
26.0
100
vitamin B12
24.0
50
22.0
0
0.0
20.0
5.0
Column:
Sample:
Eluent:
Flow rate:
System:
mAU
10.0
15.0
20.0
min
TSK-GEL Super AW3000 (Tosoh Biosciences)
10 µl (0.24% CV) protein mixture
50 mM sodium phosphate, 150 mM NaCl, pH 7.0
0.30 ml/min (64 cm/h)
ÄKTApurifier
mS/cm
ovalbumin
BSA
28.0
ribonuclease A
150
18.0
26.0
100
aprotinin
24.0
50
vitamin B12
22.0
Resolution is a function of the selectivity of the medium and the
efficiency of that medium to produce narrow peaks. Final
resolution is influenced by many factors including column
length, flow rate, bead size and pore size distribution.
In theory, efficiency can be improved by decreasing the particle
size of the medium, i.e. the 4 µm particle size of TSK-GEL Super
AW 3000 should help improve resolution compared with
Superdex 75 10/300 GL, which has a particle size of 13–15 µm.
The result we see in practice (Fig 1) nevertheless shows that in
this application, Superdex 75 10/300 GL displays a superior
separation range. The primary reason for this is the optimized
porous bead structure and pore size distribution of Superdex 75
medium. The far more compact bead structure of the polymerbased TSK-GEL column results in a much smaller accessible
pore volume in the given molecular range < 60 000. This is
seen in the poor separation, especially between
ribonuclease A and aprotinin.
In addition, column length always has an impact on resolution
for isocratic separations (in contrast to gradient techniques).
The bed height of the TSK-GEL column (15 cm) is half that of
the Superdex column and this difference also contributes to
the lower resolution of TSK-GEL Super AW 3000.
Conclusion
0
0.0
The selectivity of a gel filtration medium—the degree of
separation between peaks—depends solely on its pore size
distribution and, in a given matrix, this regulates the
separation range.
20.0
5.0
10.0
15.0
20.0
min
18.0
Fig 1. Resolution and analysis time of (A) Superdex 75 10/300 GL and (B) TSK-GEL Super
AW3000 at the same linear flow rate. The blue line is UV absorbance at 280 nm, the red
line is conductivity.
Super AW 3000 gave good separation between high and low
molecular weight proteins (BSA/ovalbumin and ribonuclease
A/aprotinin respectively), but the resolution between ribonuclease A and aprotinin was poor. In contrast, Superdex 75
10/300 GL gave a good separation across the whole range.
Note also that with the TSK-GEL column, the buffer conductivity
dipped sharply after 10 minutes elution (shows the total volume
of the column), and that vitamin B12 was retarded in the column.
This indicates that separation mechanisms other than pure gel
filtration are taking place in the TSK-GEL column, at least for
small molecules, and that this can lead to unwanted results.
Superdex 75 10/300 GL gave the highest resolution for
the test sample mixture. Its run time was 28 minutes.
TSK-GEL Super AW3000 displayed poorer resolution,
especially between ribonuclease A and aprotinin.
At 14 minutes, its run time was nevertheless faster.
The TSK-GEL column is thus better suited to rapid group
separation where there is large size difference between
molecules, rather than analytical fractionation.
To view this article online, go to
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Discovery Matters Issue 1 2005 GE Healthcare 15
Innovations Forum: Proteomics and protein analysis
Automated identification and quantitation of proteins and
peptides in mammalian samples using LC-MS/MS
A. P. Jonsson and H. Pettersen
GE Healthcare, Uppsala, Sweden
DeCyder™ MS Differential Analysis Software was used
successfully for the automated analysis of large data sets
generated from two different complex samples by LC-MS.
Data analysis was completed within a few hours, compared
with several days for manual methods.
Introduction
Differential expression analysis in LC-MS-based proteomics has
been hampered in part by the lack of suitable software tools for
data analysis. Large amounts of experimental data are easily
generated in protein and peptide profiling experiments, but
data analysis is time and labor consuming. In many cases
the analysis is manual and can take days or even weeks.
DeCyder MS Differential Analysis Software (DeCyder MS) is novel
software that integrates visualization, detection, comparison,
protein identification, and statistical tools. It simplifies the
evaluation of large LC-MS and LC-MS/MS data sets for the
relative quantitation of peptides and proteins. It supports fully
automatic detection and comparison, as well as interactive
confirmation (simultaneous view of raw data with peak
detection and charge state) of the assignments. Thus, this tool
minimizes user-to-user variation and can increase the
researcher’s productivity several-fold due to reduced analysis
time and labor.
and time-dose studies. When MS/MS data is available,
peptides that show differential expression can be submitted
for protein ID. Protein ID information can in turn be used to
look at co-variation of peptides from the same protein.
In this article, we applied DeCyder MS software to the analysis
of the following experimental models to distinguish differentially
expressed proteins:
• Mouse brain tissue with and without the enzyme
N-deacetylase/N-sulfotransferase-1 (NDST-1). This
experiment represented a control/treated model
generating a large amount of complex data.
• Rat insulinoma cell–secreted products resulting from
forskolin treatment, analyzed by high-resolution Fourier
Transform (FT)–MS. This experiment demonstrated the ability
of DeCyder MS to analyze a control/treated “peptidomics”
experiment from an FT-MS platform.
The experimental workflow is shown in Figure 1.
Results and discussion
DeCyder MS performs two main analysis procedures:
• Peptide detection. The PepDetect module provides consistent
and accurate peptide detection, background subtraction,
isotope and charge-state deconvolution, and peak volume
calculations using novel imaging algorithms. When MS/MS
data is available, all or a selected subset of peptides can be
submitted for protein identification (ID).
Control/treated experiment—mouse brain
DeCyder MS analysis of the mouse brain samples with the
presence/absence of the biosynthetic enzyme NDST-1 detected
approximately 1000 peptides (Fig 2) in each of the six intensity
maps. Matching the six intensity maps resulted in 573 matches
where p values could be calculated (i.e. data from two or more
intensity maps in each group was available). Twenty of these
had p values below 0.01, indicating significantly differentially
expressed peptides. The data was analyzed in 0.5 h, compared
with several days to weeks using conventional methods that
require total manual analysis.
• Run-to-run matching. The PepMatch module aligns peptides
from different runs. Using statistical tools, peptides that
express consistent differences among samples across
multiple runs are identified and presented together with a
level of confidence for each of those differences. Various
normalization techniques can be applied to improve the
results further. The module supports a wide range of
experimental designs, such as control/treated experiments
Control/treated experiment—rat insulinoma cells
The rat insulinoma cells were analyzed using an FT-MS, with
four sample pools from untreated cell cultures as control and
four from forskolin-stimulated cell cultures as treated. The highresolution data was successfully imported and analyzed using
DeCyder MS software. The differences in the rat insulinoma cells
with and without forskolin treatment were quantitated by
DeCyder MS (Fig 3), using ubiquitin as the internal standard. The
16 Discovery Matters Issue 1 2005 GE Healthcare
Innovations Forum: Proteomics and protein analysis
sample
sample
prep
trypsin
digestion
peptide
separation
MS/MS
DeCyder
MS analysis
Fig 1. LC-MS workflow.
Fig 3. Graphs from FT-MS data showing the insulin-15+ ion. Left: Intensity map with
detection markers (orange). Upper right: Found (yellow line) and theoretical (black line)
isotope distribution. Lower right: 3-D image (black) with detection markers (orange).
Fig 2. Intensity map showing retention time vs. m/z for a mouse brain sample.
Approximately 1000 peptides where detected in each of the six intensity maps (without
using background correction). Matching the six intensity maps resulted in 573 matches
where p values could be calculated (i.e. data from two or more intensity maps in each
group was available). Twenty of these had p < 0.01, indicating significantly differentially
expressed peptides.
upregulation of insulin was approximately 66%. The data
was analyzed in 0.5 h, compared with 1–2 days using
conventional methods.
Conclusions
Tremendous amounts of data are generated by the typical
LC-MS/MS experiment in proteomics. Teasing apart the nuances
of this data and accurately answering questions on differential
expression of proteins is a difficult task, but one that is essential
for the early phases of biomarker discovery. By using an LC
system that delivers reproducible retention times—the
Ettan MDLC—in combination with an MS/MS instrument
that provides high sensitivity and speed—such as the
Finnigan™ LTQ™ linear ion trap—the data can be analyzed
effectively by DeCyder MS software. Using this software,
differentially expressed peptides are automatically detected,
compared, quantitated, and identified.
Data generated from rat insulinoma cells with an FT-MS, more
complex than that from low-resolution platforms such as
standard ion trap mass spectrometers, was also efficiently
analyzed by DeCyder MS. The FT data analysis generally
required 1–5 min to detect a data file and < 10 s to compare
12 data files using a standard office computer. This was a
significant savings of time and labor.
The reproducible gradient formation and solvent delivery
features of Ettan MDLC provided reproducible retention
times of the eluted peptides. This improved the matching
performance of DeCyder MS because the retention time
tolerance limits could be set low, minimizing the number
of mismatches.
For more information, including experimental methods,
please see the application note Automated identification
and quantitation of proteins and peptides in mammalian
samples using LC-MS/MS (11-0027-39), available at
www.amershambiosciences.com/promo_dm004
Acknowledgements
John Flensburg, GE Healthcare, is acknowledged for supplying the LC-MS/MS data for
the mouse brain samples.
Ordering Information
DeCyder MS Differential Analysis Software
Data from mouse brain tissue, with significant variation
between groups of samples, was used to demonstrate the
utility of the DeCyder MS software. DeCyder MS automatically
processed these large, complex data sets, resulting in an
average time and labor savings of 50–75% (< 2 min to detect
a data file and < 5 s to compare 12 data files using a standard
office computer) or more depending on the type of analysis
being performed.
11-0013-32
(including PC and single concurrent network user license)
DeCyder MS Differential Analysis Software
11-0013-31
(including single concurrent network user license)
Ettan MDLC
18-1176-44
Trypsin, sequencing grade, 10 × 250 µg
17-6002-75
2-D Clean-Up Kit
80-6484-51
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Discovery Matters Issue 1 2005 GE Healthcare 17
Innovations Forum: Drug screening and cellular assays
siRNA screening of the cell cycle with
two dynamic GFP sensors
M. Kenrick, S. Hancock, S. Stubbs, and N. Thomas
GE Healthcare, The Maynard Centre, Cardiff, UK
Recent advances in siRNA methodologies and the
development of high-throughput image analysis platforms
such as the IN Cell Analyzer have revolutionized the
functional analysis of genes and proteins. Here, we describe
the application of two stable cell lines expressing green
fluorescent protein (GFP)✧ cell cycle sensors to screen a
library of siRNAs directed against key cell cycle control
genes. Imaging of GFP intensity and distribution within
these two cell lines allows cell cycle position to be assigned
by automated image analysis procedures and permits their
use in the screening of drugs that block the cell cycle.
Introduction
Advances in synthetic and virally encoded siRNA methodologies
(1, 2) have now reached a stage where large scale RNAi screens
can be applied to mammalian cells (3–5). In addition to the provision of large numbers of validated siRNAs, efficient mammalian
siRNA functional screens will require information-rich model
systems that allow abstraction of multiparameter data at a
level of throughput compatible with large-scale projects.
Fortunately, advances in the capabilities of siRNA have been
matched by the development of sophisticated fluorescence
imagers and software capable of imaging and analyzing cellular
events in live cells at high-throughput (6, 7). Such instrumentation
enables study of complex systems by combining data from
fluorescent cellular sensors with morphological parameters to
provide a detailed description of the phenotypic effects of
siRNAs in cellular screens.
In this study we have used two stable cell lines (8–10)
expressing GFP cell cycle sensors to screen a library of siRNAs
directed against key cell cycle control genes (Fig 1).
siRNA screening
Cell cycle gene knockdowns were carried out using a
Dharmacon siARRAY™ siRNA library of 112 siRNA pools each
comprising four siRNAs directed against a single cell cycle
related gene. Additional scrambled sequence and Cy™5 labeled
siRNAs were used as controls and to determine transfection
efficiency. siRNAs were transfected into G2/M cell cycle phase
18 Discovery Matters Issue 1 2005 GE Healthcare
Fig 1. G2/M and G1/S cell cycle phase marker (CCPM) constructs and cell lines. The
expression, localization and degradation of GFP fusion proteins under the control of
well characterized cell cycle control and response elements permits determination of
cell cycle position in living cells. The G2/M CCPM is expressed under the control of the
cyclin B1 promoter initiating EGFP expression in late S phase. As cells progress through
G2, cytoplasmic EGFP intensity increases and at prophase the construct localizes to the
nucleus. GFP intensity reaches a maximum at mitosis, and is then rapidly degraded
under control of the cyclin B1 destruction box (D-box) such that the two resulting
daughter cells in G1 are nonfluorescent. The G1/S CCPM is expressed constitutively by
the ubiquitin C promoter giving low level fusion protein expression throughout the cell
cycle. In G1 cells the fusion protein is strongly localized to the nucleus and on transition
to S phase phosphorylation of the PSLD domain leads to export to the cytoplasm,
which continues through G2 phase completely reversing the nuclear/cytoplasmic
distribution of the fusion protein.
marker (CCPM) and G1/S CCPM expressing U2OS cells. Details
are available in the online version of this article.
Cellular imaging was performed on an IN Cell Analyzer 1000
using a 20× objective and 475BP20/535BP50 (GFP) and
620BP60/700BP75 (DRAQ5) excitation/emission filters. Image
stacks were converted to IN Cell Analyzer 3000 format and the
resulting images analyzed for cell number, cell cycle distribution,
and morphology.
Results
Analysis of G1/S CCPM cells allowed the effects of cyclin E
knockdown on G1 to S phase transition to be quantitated (Fig 2).
Control G1/S CCPM cells showed the same proportion (76%)
of cells in G1 or S phase as control G2/M CCPM cells (74%),
which was resolvable in G1/S CCPM cells to 9% G1 cells and
✧
See licensing information on page 24.
Innovations Forum: Drug screening and cellular assays
6000
C
PLK
Granular DNA fluorescence
5000
Knockdown of polo-like kinase (PLK) with siRNA has been
previously shown to inhibit cell proliferation, arrest cells in
mitosis, and induce apoptosis (11). Cell cycle analysis of G2/M
CCPM cells treated with siRNA directed against PLK showed a
dramatic increase in mitotic cells 48 h after transfection with
50 nM siRNA (Fig 3B). Extreme sensitivity to PLK knockdown was
confirmed by analysis of G1/S CCPM (Fig 3A) and G2/M CCPM
(data not shown) 24 h following transfection with 5 nM siRNA,
which showed an increase in G2 and M phase cells and a
corresponding decrease in G1 cells.
Re-analysis of all image data from the siRNA library screen
using DNA granularity revealed that PLK siRNA gave the most
significant induction of apoptosis across the target genes in this
study (Fig 3C).
Conclusions
Perturbation of sensitive and dynamic phenotypic cellular
assays via siRNA provides a powerful tool for functional analysis
of the cell cycle. High-throughput subcellular imaging and
automated multiparameter image analysis provides an
information-rich environment to screen and study effects of
gene knockdown with siRNA.
References
1. Kumar, R. Conklin DS, Mittal V. High-throughput selection of effective RNAi probes for
gene silencing. Genome Res. 13 (10), 2333–2340 (2003).
2. Luo, B. et al. Small interfering RNA production by enzymatic engineering of DNA
(SPEED). Proc. Natl. Acad. Sci. USA 101 (15), 5494–5499 (2004).
3000
2000
1000
Fig 2. Cyclin E siRNA blocks G1 to S phase transition. G1/S CCPM cells were transfected
with an siRNA pool directed against cyclin E. Following culture for 24 h, cells were
pulsed with BrdU for 1 h, fixed and BrdU incorporation detected with the Cell
Proliferation Fluorescence Assay. G1 and S phase cells were quantitated using object
intensity image analysis to identify cells with green (G1) and red (S) nuclei.
67% S phase cells. On knockdown of cyclin E the proportion of
cells in G1 or S phase remained constant at 76%, as observed
with G2/M CCPM cells. However the balance between the two
phases shifted significantly to 27% G1 cells and 49% S phase
cells, reflecting the critical role of cyclin E in G1 to S transition.
4000
0
siRNA
Fig 3. Cell cycle arrest and induction of apoptosis by PLK siRNA. (A) G1/S CCPM cells
transfected with 5 nM PLK siRNA and pulsed with BrdU after 24 h. (B) G2/M CCPM cells
transfected with 50 nm PLK siRNA and incubated for 48 h. (C) Measurement of DNA
granularity as a measure of apoptosis for all siRNAs used.
3. Berns, K. et al. A large-scale RNAi screen in human cells identifies new components of
the p53 pathway. Nature 428 (6981), 431–437 (2004).
4. Paddison, P.J. et al. A resource for large-scale RNA-interference-based screens in
mammals. Nature 428 (6981), 427–431 (2004).
5. Mousses, S. et al. RNAi microarray analysis in cultured mammalian cells. Genome
Res. 13 (10), 2341–2347 (2003).
6. Ramm, P. and Thomas, N. Image-based screening of signal transduction assays.
Sci. STKE. (177), (2003).
7. Price, J.H. et al. Advances in molecular labeling, high-throughput imaging and
machine intelligence portend powerful functional cellular biochemistry tools.
J. Cell. Biochem. Suppl. 39,194–210 (2002).
8. Thomas, N. Lighting the circle of life: fluorescent sensors for covert surveillance of the
cell cycle. Cell Cycle 2 (6), 545–549 (2003).
9. Thomas, N. et al. Characterization and gene expression profiling of a stable cell line
expressing a cell cycle GFP sensor. Cell Cycle 4 (1), 191–195 (2005).
10. Gu, J. et al. Cell cycle-dependent regulation of a human DNA helicase that localizes
in DNA damage foci. Mol. Biol. Cell. (7), 3320–3332 (2004).
11. Liu, X. and Erikson, R.L. Polo-like kinase (Plk)1 depletion induces apoptosis in cancer
cells. Proc. Natl. Acad. Sci. USA 100 (10), 5789–5794 (2003).
For a more detailed version of this article, please visit
www.amershambiosciences.com/promo_dm006
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IN Cell Analyzer 1000
25-8010-26
IN Cell Analyzer 3000
25-8010-11
Cell Proliferation Fluorescence Assay
25-9001-89
(500 assays)
G2M Cell Cycle Phase Marker Assay,
25-8010-52
6 month assay evaluation (3 vials)
G2M Cell Cycle Phase Marker Assay,
25-9002-55
nonprofit research (3 vials)
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Discovery Matters Issue 1 2005 GE Healthcare 19
Innovations Forum: Drug screening and cellular assays
SPA Imaging Charge-Based Binding Beads: A different
approach to the capture of assay product
D. J. Powell and M. J. Price-Jones
GE Healthcare, the Maynard Centre, Cardiff, UK
Introduction
In a bead-based technology such as SPA imaging, specific
signal is generated by the capture of assay product onto the
bead, thus bringing the radiolabel into close proximity with the
scintillant contained within the bead. Typically, SPA imaging
assays make use of well-characterized interactions such as
biotin-streptavidin and the capture of cell membrane glycoprotein by wheat germ agglutinin (WGA). However, this is not
possible with all assay substrates.
Many substrates exist as glutathione S-transferase (GST) fusion
proteins, which are not generally amenable to capture using
WGA, and biotinylation is usually not an option. SPA Imaging
Charge-Based Binding Beads offer an opportunity to use
GST fusion substrates without the need for modification or
additional reagents.
To demonstrate the utility of SPA Imaging Charge-Based Binding
Beads, an assay was developed for the c-Jun N-terminal kinase
JNK3 (SAPK1b). This kinase has been implicated in the
pathogenesis of several neurological disorders, including
Alzheimer’s disease, Parkinson’s disease, and stroke (1).
Assay development
SPA imaging assay development requires several stages
including selection of reagent masses for signal optimization,
bead mass, buffer components, and, in the case of enzyme
assays, stop reagent. The order in which these stages are
performed is not critical.
Stop reagent
Because this assay is using a charge-based approach to
capture the assay product, selection of the stop reagent is one
of the most important steps. With the wrong components, stop
reagent could actually inhibit capture of the product. For kinase
assays, another issue is the presence of unincorporated
20 Discovery Matters Issue 1 2005 GE Healthcare
[γ-33P]ATP, which could also potentially bind to the bead and
cause significant non-specific binding (NSB).
Two different stop buffers were assessed: 50 mM sodium
acetate, pH 5.0, 100 mM ATP; and 10 mM phosphoric acid,
0.1% (v/v) Triton™ X-100, 50 mM ATP.
The phosphoric acid-based stop mix showed a marked
advantage over the sodium acetate-based stop mix in terms
of signal to noise and also in total signal (not shown). Although
there was a decline in signal over time, as evidenced by the
decline in signal to noise (Fig 1), it always remained well in
excess of the sodium acetate equivalent. Use of appropriate
controls on each plate prevents this becoming an issue of
assay variation.
4.5
Sodium acetate
Phosphoric acid
4.0
Signal:Noise
Cloning and purification of functionally active target
proteins is frequently accomplished using the glutathione
S-transferase (GST) system. Here we describe the development and validation of a SPA enzyme assay based on the
use of SPA Imaging Charge-Based Binding Beads with a
GST fusion substrate for the capture of assay product.
3.5
3.0
2.5
2.0
1.5
0
5
10
15
Time (h)
20
25
30
Fig 1. Effect of stop
reagent on signal to
noise ratio (S:N) over
time. To eliminate any
variation due to wellto-well dispensing, the
plus- and minus-enzyme
reactions were incubated
in bulk in microcentrifuge
tubes before aliquots
were transferred to a
384-well non-binding
surace plate (Corning) for
addition of stop reagent
and then imaging.
Bead mass
The amount of bead used in an assay has a bearing on factors
such as cost and performance. Too much bead adds cost per
well whereas too little bead may harm assay performance.
Initially, a 100 µg bead mass was chosen to be able to test
reagents and optimize other assay components. The effect of
varying the amount of bead was then assessed.
The data shows (Fig 2) that once the bead mass increased
beyond 100 µg, the signal increased, but only with a
concomitant rise in background (no enzyme).
The data in Table 1 shows that 100 µg of bead offered the best
signal-to-noise ratio. Extra bead did increase the total signal, but
only at the cost of increased background.
Innovations Forum: Drug screening and cellular assays
1500
+ Enzyme
- Enzyme
1200
IOD
900
600
300
0
0
50
100
150
200
250
Bead mass (µg)
50
3.02
3.74
150
2.86
200
2.73
250
2.64
To further validate the assay, the JNK selective inhibitor
SP600125 (Calbiochem) was tested. This inhibitor shows
> 20-fold selectivity for the JNK family of kinases (2).
Assay conditions used for the inhibitor curve were broadly as
used for the time course experiment, except that DMSO was
included to a final concentration of 4% (v/v) to keep the inhibitor
in solution.
Under these conditions, the IC50 for SP600125 was 0.2748 µM
(Fig 4). Literature values for inhibition of JNK3 by SP600125 vary
from 0.09 µM (2) to 0.19 µM (1). Variations are most likely due to
variations in the ATP concentrations used.
Table 1. Signal to noise ratios (S:N) at varying
bead masses
S:N
100
Assay validation
120
100
% Inhibition
Bead mass (mg)
Fig 2. Effect of bead
mass on assay
performance (n = 3,
±SEM). Bulk reaction
mixes were set up to
eliminate any well-towell variation due to
incubation or dispensing.
Following incubation,
reaction equivalents
(25 µl) were dispensed
to 384-well plates and
stopped by the addition
of the phosphoric acid
stop mix, which
contained varying
amounts of bead in the
range 50–250 µg.
80
60
40
20
0
Time course experiment
-3
-2
-1
The assay was linear for around 15 min; reaching a plateau
after 30 min (Fig 3). Based on these results, a 15 min incubation
time was chosen as standard.
Assay conditions:
Enzyme:
Substrate:
ATP:
[γ-33P]ATP:
Assay volume:
Assay buffer:
10 ng JNK3/well
0.5 µg GST-c-Jun (1–169)/well
4 µM
0.1 µCi/well
25 µl
25 mM Tris-HCl (pH 7.5),
5 mM β-glycerophosphate,
2 mM DTT, 0.1 mM sodium
orthovanadate, 10 mM MgCl2
50 µl/well
Stop mix:
(bead in buffer)
600
Fig 3. JNK3 time course
(n = 3, ±SEM). Assays
were performed at 30 ˚C.
0
1
[SP600125] µM
With key conditions established using bulk reaction mixes, the
assay was moved to a plate-based format. This format was
then used to establish assay performance over time.
2
3
Fig 4. SP600125
inhibition of JNK3
activity. JNK3 was preincubated with SP600125
for 10 min prior to the
addition of the remaining
assay components.
Assay incubation was
15 min before the
addition of stop reagent.
The beads were allowed
to settle for 1 h before
imaging. Data shown is
single point, averaged
from triplicate raw data
points. The IC50 for
SP600125 was determined to be 0.2748 µM
(95% confidence limits
0.1618 to 0.4668 µM).
Conclusion
SPA Imaging Charge-Based Binding Beads have been shown to
be suitable for the assay of kinase activity using GST fusion
substrates.
References
1. Resnick, L. and Fennell, M. Targeting JNK3 for the treatment of neurodegenerative
disorders. Drug Disc Today 9(21), 932–939 (2004).
2. Bennett, B.L. et al. SP600125, an anthapyrazolone inhibitor of Jun N-terminal kinase.
Proc. Natl. Acad. Sci. USA 98(24), 13681–13686 (2001).
To view this article online, please visit
www.amershambiosciences.com/promo_dm007
500
IOD
400
300
200
Ordering Information
100
SPA Imaging Charge-Based
0
0
10
20
30
Time (min)
40
50
RPNQ0320
Binding Beads (500 mg)
To shop online, go to www.amershambiosciences.com
Discovery Matters Issue 1 2005 GE Healthcare 21
technical tips
Ettan DIGE: New protocol for selective labeling of cell
surface proteins using CyDye DIGE Fluor minimal dyes
Introduction
Cell surface proteins, partly due to their low abundance (1–2%
of cellular proteins), can be difficult to detect using 2-D electrophoresis without fractionation or some other type of enrichment.
These proteins are often poorly represented in 2-D gels due to
their hydrophobic nature and high molecular weight (1).
Here we outline a new protocol using CyDye™ DIGE Fluor
minimal dyes✧ to visually enrich for and detect this group
of proteins (Fig 1).
1. Cell-surface protein labeling protocol
Cy5
Cy5
cell
Fractionated
sample
cell
+ CyDye
in 200 µl ice cold labeling buffer (HBSS pH 8.5, 1M urea). Cells are
labeled with 600 pmol CyDye DIGE Fluor minimal dyes for
20 min on ice in the dark. The reaction is quenched by adding
20 µl of 10 mM lysine and incubated for 10 min. The surfacelabeled cells are washed twice by resuspension in 500 µl HBSS
pH 7.4 followed by centrifuging at 800 × g at 4 ˚C for 2 min.
The cell surface protein labeling protocol was compared with
the standard Ettan DIGE protocol using CHO-K1 cells (Fig 2).
DeCyder 2-D software was used to evaluate the results. This
comparison revealed over 80 novel proteins spots present only
in the cell surface protein labeled fraction at a ratio of more
than 10. The new protocol also improved the detection of
several proteins when compared with the standard method.
2-D DIGE
Cells lysed
Cy5
Fig 2. Two identical
samples from CHO-K1
cells grown in the same
flask were labeled in
parallel using the two
different protocols.
Samples from the two
protocols were run in the
same 2-D gel. Red spots
represent proteins
labeled with CyDye DIGE
Fluor Cy5 minimal dye
(RPK0275) using the cell
surface labeling protocol.
Green spots represent
proteins labeled with
CyDye DIGE Fluor Cy3
minimal dye (RPK0273)
using the standard
protocol. Yellow spots
represent proteins
present in both samples.
Non-fractionated
sample
2. Standard Ettan DIGE protocol
cell
Cells lysed
+ CyDye
Fractionated
sample
2-D DIGE
Fig 1. Overview of the two labeling workflows.
This protocol is rapid, simple to use, and all three CyDye DIGE
Fluor minimal dyes (Cy™2, Cy3 and Cy5) can be used to label cell
surface proteins similarly. This allows for multiplexing according
to 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE)
using Ettan™ DIGE technology and analysis of protein expression changes using DeCyder™ 2-D Differential Analysis Software.
Small changes in abundance can be detected with high accuracy and results are supported by defined statistical methods.
Cell surface labeling protocol
Adherent cells are detached non-enzymatically, counted,
and divided into aliquots of 5–10 × 106 cells. Cells growing in
suspension are simply counted and aliquotted. Cells are pelleted
(800 × g for 5 min) and the supernatant removed. Pellets are
washed by resuspension in 1 ml ice cold Hank’s Balanced Salt
Solution (HBSS) pH 8.5 and centrifuged at 800 × g at 4 ˚C for
2 min. The supernatant is removed and the pellet resuspended
22 Discovery Matters Issue 1 2005 GE Healthcare
For a detailed description of this comparison, please see the
application note Selective labeling of cell surface proteins
using CyDye DIGE Fluor minimal dyes (11-0033-92),
available at www.amershambiosciences.com/promo_dm009
References
1. Shin, B.K. et al. Global profiling of the cell surface proteome of cancer cells uncovers
an abundance of proteins with chaperone function. J Biol Chem. 9, 7607–7616 (2003).
Acknowledgements
We thank Professor Dontscho Kerjaschki, Corina Mayrhofer and Sigurd Krieger at the
Institute of Clinical Pathology, University of Vienna, Austria for their collaboration.
✧
See licensing information on page 24.
technical tips
Gravity-flow purification of histidine-tagged proteins using
Ni Sepharose 6 Fast Flow packed in PD-10 columns
A protocol for gravity-flow purification of a histidine-tagged
green fluorescent protein (GFP-[His6]) using Ni Sepharose™ 6
Fast Flow packed in Empty Disposable PD-10 Columns is
described✧. Using this protocol, the purification performance of
Ni Sepharose 6 Fast Flow was compared with the performance
of Ni-NTA Superflow™ and Ni-NTA Agarose (both Qiagen Inc.).
A
Mr
(× 10-3)
M
flow wash
through 1
wash elution elution elution
2
1
2
3
M
flow wash
through 1
wash elution elution elution elution
2
1
2
3
4
M
flow wash
through 1
wash elution elution elution elution
2
1
2
3
4
97
66
Sample binding
45
Four milliliters of clarified E. coli lysate (about 9 mg total GFP[His]6) was added to 1 ml of a 50% slurry of each media. Sample
and media were incubated at room temperature on a shaker at
low speed for 1 h.
30
20.1
14.4
Buffer wash and elution
Samples and media were loaded onto the PD-10 columns and
the flowthrough was collected. Columns were washed using
4 bed volumes (BV; 2 ml) binding buffer (20 mM sodium
phosphate, 500 mM NaCl, 20 mM imidazole, pH 7.3). Wash
fractions were collected. Elution was performed using
approximately 5 BV (2.5 ml) of elution buffer (20 mM sodium
phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.3).
Absorbance was measured at 280 nm (Table 1) and the purity of
pooled fractions was analyzed by SDS-PAGE.
B
Mr
(× 10-3)
97
66
45
30
20.1
Table 1. Comparison of three media used for the purification of
histidine-tagged proteins.
Medium
(0.5 ml in slurry)
14.4
Eluted pool (A280)
Approx. amount of
GFP-(His)6 eluted (mg)
Yield of
GFP-(His)6 (%)
Ni Sepharose 6 Fast Flow
0.437
6.6
73
Ni-NTA Superflow
0.281
4.2
46
Ni-NTA Agarose
0.283
4.2
46
C
Mr
(× 10-3)
97
66
Results
Using 0.5 ml Ni Sepharose 6 Fast Flow allowed binding of 73% of
the applied GFP-(His)6 whereas the two Ni-NTA media bound
only 46%. Binding to Ni Sepharose 6 Fast Flow was 1.6-fold
greater than binding to the Ni-NTA media. Purity of GFP-(His)6
obtained from runs on the three media was similar as
determined by SDS-PAGE (Fig 1).
45
30
20.1
14.4
Fig 1. Purity of GFP-(His)6 purified by batch/gravity-flow on 0.5 ml of media.
Electophoresis was performed using PhastGel™ Gradient 10–15 gels.
(A) Ni Sepharose 6 Fast Flow, (B) Ni-NTA Superflow, and (C) Ni-NTA Agarose.
The complete protocol can be found at
www.amershambiosciences.com/protocol-his.
✧
See licensing information on page 24.
Discovery Matters Issue 1 2005 GE Healthcare 23
General Electric Company reserves the right, subject to any regulatory approval if
required, to make changes in specifications and features shown herein, or discontinue
the product described at any time without notice or obligation. Contact your GE
Representative for the most current information.
© 2005 General Electric Company—All rights reserved. Printed in the UK.
GE and GE Monogram are trademarks of General Electric Company. Advantage
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Superdex, Tricorn, and UNICORN are trademarks of GE Healthcare Limited.
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siArray is a trademark of Dharmacon Inc.
Sypro is a trademark of Molecular Probes Inc.
Superflow is a trademark of Sterogene Bioseparations Inc.
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