Find differences in protein expression with Ettan DIGE

Find differences in protein expression with Ettan DIGE
GE Healthcare
Find differences
in protein expression
with Ettan DIGE
2-D Fluorescence Difference Gel Electrophoresis
imagination at work
Results you can trust in
protein abundance analysis
Ettan DIGE delivers these important benefits:
Ettan™ DIGE system uses 2-D Fluorescence Difference
Gel Electrophoresis (2-D DIGE), the benchmark for
protein abundance analysis. The world’s leading
pharmaceutical and academic centers of excellence
are increasingly switching to Ettan DIGE because of
the unrivaled accuracy it provides.
• Standardization: uses a pooled internal standard
to deliver the lowest possible gel-to-gel variation
• Accuracy: ensures that the smallest possible real
differences in protein abundance are detected
with unparalleled statistical confidence
Standardize your 2-D results
Ettan DIGE uses multiplexing, the simultaneous coseparation of multiple, fluorescently labeled samples,
including a pooled internal standard on each gel.
Each protein spot has its own internal standard,
which ensures that the differences you see in protein
abundance are real. This is the only effective way
to minimize gel-to-gel variation and significantly
increase accuracy and reproducibility.
• Reproducibility: leads to consistent and
comparable conclusions
• Cost effectiveness: achieves dependable results
with far fewer gels
• Efficiency: increases throughput and significantly
reduces analysis time
Efficiency saves time and costs
The level of reproducibility and statistical confidence
with Ettan DIGE ensures that you get results you can
rely on. You can also be confident that you’re not
missing important differences in protein abundance.
Because Ettan DIGE requires far fewer gels than
other methods for measuring protein abundance
differences, it also provides significant time and cost
savings.
• Proven method: has rapidly become established
in leading laboratories worldwide
2
Ettan DIGE: raising the standard of 2-D electrophoresis
3
Conclusions you can defend
Accurate results lead to confident conclusions and the right
decisions. In proteomics research, you need to be sure you
are choosing the right proteins to select, characterize, and
quantitate. Ettan DIGE minimizes the danger of missing vital
biomarkers, so you can be confident you have selected the
right candidates.
Ettan DIGE is a proven method that combines the most
statistically accurate protein abundance data with the
greatest resolving power available in 2-D electrophoresis.
Whether you’re interested in researching disease, identifying
biomarkers, or understanding tissue differentiation, Ettan
DIGE gives you findings you can believe in, which lead to
conclusions you can defend.
“Without the benefit of the internal standard, 42 of these
proteins would have been overlooked due to variation between
normal and tumor samples ... compared with individual DIGE
comparisons made within a single 2-D gel.”
Friedman, D. B. et al. Proteome analysis of human colon cancer by two-dimensional difference gel electrophoresis and mass spectrometry.
Proteomics 4, 793–811 (2004).
4
The use of a pooled internal standard leads to accurate
conclusions
Traditional 2-D electrophoresis
Four different samples run on four different gels
Sample 1
Sample 2
The abundance of this particular protein
spot appears to be increasing in samples
3 and 4. Is this increase due to system
variation or induced biological change?
4
3
2
1
0
1
2
3
4
Experimental conclusion
Sample 3
Sample 4
Without running a significant number
of replicates to average the results,
the conclusion would be an increase in
abundance in samples 3 and 4.
Ettan DIGE using a pooled internal standard
Four different samples, plus one internal standard, on two different gels
Internal standard (Cy2)
Gel A
Sample 1 (Cy3)
4
4
3
3
2
2
1
1
0
Internal standard (Cy2)
Sample 2 (Cy5)
1
2
3
4
Sample 3 (Cy3)
Sample 4 (Cy5)
Gel B
5
0
1
2
3
4
Experimental conclusion
The same internal standard is run on
both gels. The increase in abundance of
protein in gel B, as shown by the increase
in the internal standard, is due to gel-to-gel
variation. When the internal standard is
normalized between gels A and B, the
conclusion is that the abundance of
protein in sample 3 has actually decreased.
The differences you see in
protein abundance are real
Ettan DIGE is the only system with a pooled internal
standard for every spot on every gel. Ettan DIGE uses
prelabeling of protein samples with size- and chargematched, spectrally resolvable CyDye™ DIGE Fluors.
Differences in protein abundance are then accurately
quantitated using DeCyder™ 2-D Differential Analysis
Software.
This dedicated 2-D analysis software uses the internal
standard to derive data from within gels and then between
gels, minimizing gel-to-gel variation. This allows you to
achieve detection of < 10% differences between samples
with > 95% statistical confidence, within minutes.
After separation on Ettan IPGphor™ or Multiphor™ II IEF
system in the first dimension and Ettan DALTtwelve or Ettan
DALTsix Large Vertical Electrophoresis System in the second
dimension, samples are scanned with Typhoon™ Variable
Mode Imager or Ettan DIGE Imager.
“Ettan DIGE with the internal standard delivers
reliable results by decreasing gel-to-gel variation
more than ever before.”
Dr. Jörgen Östling, AstraZeneca R&D, Mölndal, Sweden
Ettan DIGE performance comparison*
Matched spots
Mean CV%
DIGE dyes, no internal standard, without DeCyder 2-D software
1300
26%
DIGE dyes, with internal standard, without DeCyder 2-D software
1300
15%
Ettan DIGE, with DeCyder 2-D Differential Analysis Software
1300
3-6%
Classical 2-D with fluorescent stain, without DeCyder 2-D software
1670
21%
* For each method, the same sample was analyzed in replicates of six gels.
6
Ettan DIGE is the only system with a pooled internal standard
for every spot on every gel
Pooled internal
standard labeled
with Cy2
Protein extract 1
labeled with Cy3
Separate
by 2-D PAGE
Image
analysis:overlay
images
Protein extract 2
labeled with Cy5
Mix labeled extracts 2-D electrophoresis
separation 7
Cy2
Excitation
wavelength
Image analysis:
data quantitation
Cy3
Excitation
wavelength
Image analysis:
data quantitation
Cy5
Excitation
wavelength
Image analysis:
data quantitation
Typhoon
Variable Mode Imager or Ettan DIGE Imager DeCyder 2-D
Differential
Analysis Software
1. Sample labeling
CyDye DIGE Fluors
Designed specifically for Ettan DIGE,
these dyes are size- and chargematched, as well as spectrally distinct,
offering bright and intense colors
with narrow excitation and emission
bands. This allows co-separation of
different CyDye DIGE fluor-labeled
samples in the same gel and ensures
that all samples are subject to exactly
the same first- and second-dimension
electrophoresis running conditions.
This limits experimental variation
and ensures accurate spot matching
within gels.
CyDye DIGE Fluor Labeling Kit
for scarce samples
These CyDye fluors label the amino
acid cysteine and are recommended
for use when scarce amounts of
sample are available (e.g., for analysis
of proteins obtained from lasercapture microdissection). Each
labeling reaction requires only
5 µg of protein sample.
CyDye DIGE Fluor minimal dyes
These CyDye fluors label the amino
acid lysine. They are the most
commonly used dyes for general
2-D electrophoresis applications
and are suitable for all sample types.
Each labeling reaction requires
50 µg of protein sample.
8
2. Sample separation:
first dimension
Ettan IPGphor 3 and Multiphor II IEF
systems
Ettan IPGphor 3 IEF allows for
high throughput first dimension
electrophoresis with increased speed,
accuracy and reproducibility. With
Ettan IPGphor Manifold or Ettan
IPGphor Manifold Light, up to 12 IPG
strips can be run simultaneously
which simplifies handling of multiple
samples and saves time. Ettan IPGphor
3 Control Software helps you manage
up to four Ettan IPGphor 3 units
simultaneously.
To perform first dimension separation
on the Multiphor II Electrophoresis
System, use the Immobiline™
Drystrip Kit. To increase efficiency
and reproducibility even further, the
specially designed IPGbox can be
IPGbox is designed for
rehydration of up to
12 Immobiline DryStrip
Gels.
Ettan IPGphor 3 system, optimized
for speed, reliability and use with
fluorescently-labeled protein samples.
9
used to rehydrate up to 12 IPG strips
independently and at the same time.
With the IPGbox, you can load samples
during rehydration by including them in
the buffer, or by using sample cups.
3. Sample separation:
second dimension
Ettan Dalt Electrophoresis System
In just 4-5 hours, Ettan DALTsix Large
Vertical System can run up to six large
labcast or precast gels (26 x 20 cm) in
the second dimension. The gels are
compatible with first dimension 18 or
24 cm Immobiline DryStrip Gels (IPG
strips) from both Ettan IPGphor and
Multiphor II IEF systems. For increased
throughput, use the EttanDALTtwelve
electrophoresis system which can
simultaneously run up to 12 largeformat gels. Ettan DALTtwelve used
with 24 cm IPG strips provides very
high resolution and maximizes the
number of distinct, quantifiable protein
spots per gel.
Choosing a product with the DIGEapproved seal assures that you will
obtain optimum results with a product
fully compatible with Ettan DIGE
technology. From sample preparation
to data analysis, there is a DIGEapproved product for every step. Look
for the DIGE-approved seal for 2-D
results you can trust.
Compact Ettan DALTsix
Large Vertical System
Ettan DALTtwelve provides high resolution,
throughput, and batch-to-batch reproducibility.
10
4. Image acquisition
Typhoon Variable Mode Imager
The Typhoon Variable Mode Imager is
a highly sensitive, variable mode gel
imager optimized to image CyDye DIGE
fluor–labeled proteins.
• Outstanding linearity, quantitative
accuracy, and extremely low limits
of detection
• Detection of small differences with
highest statistical confidence
• Consistent point light illumination
• Simultaneous imaging of two
large-format Ettan DALT gels
automatically
Ettan DIGE Imager
Ettan DIGE Imager represents leadingedge technology for generating highquality, low-noise images from your
2-D DIGE experiments. By combining
very high resolution with precise
motion control, Ettan DIGE Imager
produces accurate, multichannel
images of your Cy™2-, Cy3-, and
Cy5-labeled gel.
Gels are scanned between glass plates,
preventing drying and shrinkage and allowing
further running and rescanning, if required.
• High sensitivity for imaging
faint spots
• Software-selectable wavelengths
for better sensitivity and reduced
background
• Adjustable scan time to optimize
sensitivity
• Sealed environment for scanning
and protecting wet samples
Gels are mounted in cassettes to avoid drying.
11
5. Image analysis
DeCyder 2-D Differential Analysis Software
DeCyder 2-D software has been specifically developed to
exploit the benefits of prelabeled, multiplexed samples and
the internal standard. It automatically locates and analyzes
multiple samples in a gel and then enables comparative
analysis of multiple gels, producing accurate measurements
of differential changes in protein abundance. The software
provides statistical confidence and minimal user-to-user
variation, which reduces hands-on analysis time to minutes.
12
6. M
ultivariate analysis
in proteomics
DeCyder Extended Data Analysis (EDA) Software
DeCyder EDA is a powerful analysis tool extending the
statistical options offered in 2-D DIGE. It enables you to
switch seamlessly between EDA statistical results and
DeCyder 2-D visualization data, putting your results into
biological context by linking to internal and external
databases. Together they create a powerful platform of
image analysis tools, which offers flexibility, versatility, and
adaptability for protein profiling.
DeCyder EDA provides advanced statistical analysis in a
simple-to-use format, uncovering patterns in expression
data and relationships using multivariate analysis and
sophisticated clustering methods.
Hierarchical cluster analysis
The software helps you understand regulatory pathways,
find proteins with similar expression profiles, or group your
samples according to common expression patterns. It also
supports the identification of proteins that discriminate
between disease stages, tumor types or other sample
subtypes, giving your Ettan DIGE results clarity and
biological relevance.
K-means analysis
Principal component analysis (PCA)
13
Proven in practice
CyDye DIGE Fluor minimal dyes for tracking membrane protein abundance in epilepsy-induced rats
The results of this proteomics study demonstrate that, as part of the Ettan DIGE platform, CyDye DIGE
Fluor minimal dyes are applicable to the analysis of membrane proteins and are beneficial for use within
preclinical and clinical proteomics.
The study was carried out in collaboration with Dr. S. Hattori et al., Division of Cell Proteomics,
Institute of Medical Science, University of Tokyo, Japan.
Some examples of the proteins identified by mass spectrometry
Protein name
Differential expression*
NADH dehydrogenase alpha subunit 10
+ 6.27
Oxygen regulated protein
+ 2.10
ATP-dependent zinc metalloprotease
+ 1.96
Golgi coiled coli protein
+ 1.72
Neural adhesion molecular F3
+ 1.96
Aldehyde dehydrogenase
+ 1.67
Huntingtin interaction protein 1 related protein
+ 4.36
T-cell activation Rho GTPase-activation protein
+ 1.51
Growth factor receptor bound protein
+ 2.34
Androgen-induced prostate proliferative shutoff associated protein As3
+ 2.02
NADH dehydrogenase alpha subunit 10
- 33.02
FERM, RhoGEF and pleckstrin domain protein 2
- 1.67
Heat-stable enterotoxin receptor precursor
- 1.87
ATP synthase beta subunit
- 2.67
F1-ATPase beta subunit
- 2.17
Huntingtin interacting protein related
- 2.63
H+transporting two sector ATPase
- 1.87
* Abundance in treated sample vs. control, measured by volume ratio
14
Analyzing differences in membrane protein expression
in rat brain with 2-D DIGE
Control
Kainate
Control
Volume ratio
-33.02
Kainate
Volume ratio
+6.27
Protein spots showing a predominantly red or green color demonstrate changes in abundance between normal and kainic acid treatment (used to induce
epilepsy in rats). The centrally positioned pair of green and red spots shown above were identified as the same protein, NADH dehydrogenase alpha
subcomplex 10–like protein. This protein has undergone some structural changes caused by the kainic acid treatment.
Without 2-D DIGE
1228
PPM
CNT
SSP 9507
Without the 2-D DIGE system, several replicates must be run due to high gel-to-gel variation.
This is time-consuming,
delivering lower quality data and leading to inaccurate conclusions.
533
PPM
Courtesy
CNT of Jörgen Östling, AstraZeneca R&D Mölndal, Sweden.
15
SSP 9510
Six replicate gels
Courtesy of Jörgen Östling, AstraZeneca R&D Mölndal, Sweden
Ordering information
Product
Quantity
Sample labeling
DIGE Trial Pack, incl. CyDye DIGE Fluor minimal dye
labeling kit + 1 month free DeCyder 2-D trial license
CyDye DIGE Fluor minimal dye labeling kit CyDye DIGE Fluor, minimal labeling kit CyDye DIGE Fluor Labeling Kit for Scarce Samples
CyDye DIGE Fluor Labeling Kit for Scarce Samples
and Preparative Gel Labeling
Code no.
2 nmol
2 nmol 5 nmol 1
28-9373-73
28-9345-30
25-8010-65
25-8009-83
1
25-8009-84
Sample separation, first dimension
DeStreak Rehydration Solution 5 × 3 ml 17-6003-19
DeStreak Reagent 1 ml
17-6003-18
Ettan IPGphor 3 Isoelectric Focusing Unit
1
11-0033-64
Immobiline DryStrip Kit
1
18-1004-30
IPGbox 1 IPGbox + IPGbox Kit
28-9334-65
IPGbox Kit 10 Reswell Trays + IPGbox Insert 28-9334-92
IPG Buffer pH 5.5-6.7 1 ml 17-6002-06
IPG Buffer pH 4-7 1 ml 17-6000-86
IPG Buffer pH 6-11 1 ml 17-6001-78
IPG Buffer pH 7-11 NL 1 ml 17-6004-39
IPG Buffer pH 3-10 NL 1 ml 17-6000-88
IPG Buffer pH 3-10 1 ml 17-6000-87
IPG Buffer pH 3-11 NL 1 ml 17-6004-40
Ettan IPGphor Manifold, Complete 1
80-6498-38
Ettan IPGphor Manifold, Light Complete
1
11-0026-88
Multiphor II Electrophoresis System 1
18-1018-06
Immobiline DryStrip Gels for IEF1
pH range
Quantity
Code No
18 cm
24 cm
3.5–4.5
3–7 NL
4–7
6–9
6–11
3–10
3–10 NL 12/pack
12/pack
12/pack
12/pack
12/pack
12/pack
12/pack
–
–
17-1233-01
17-6001-88 17-6001-97
17-1234-01
17-1235-01
For local office contact information, visit,
www.gelifesciences.com/contact
GE Healthcare Bio-Sciences AB
Björkgatan 30
751 84 Uppsala
Sweden
www.gelifesciences.com/dige
17-6002-38
17-6002-43
17-6002-46
17-6002-47
–
17-6002-44
17-6002-45
Code No
pH range
Quantity
18 cm
24 cm
3–5.6 NL
5.3–6.5 6.2–7.5 7–11 NL
3–11 NL
12/pack
12/pack
12/pack
12/pack
12/pack
17-6003-56
17-6003-61
17-6003-66 17-6003-71
17-6003-76 17-6003-57
17-6003-62
17-6003-67
17-6003-72
17-6003-77
IPG strips are also available in 7, 11 and 13 cm strip lengths. For information, contact your local
GE Healthcare representative.
1
Product
Quantity
Sample separation, second dimension
Ettan DALTtwelve Separation Unit and Power
Supply/Control Unit, 115V
Ettan DALTtwelve Separation Unit and Power
Supply/Control Unit, 230V
DALTtwelve Gel Caster, complete Ettan DALTsix Electrophoresis Unit, 115V Ettan DALTsix Electrophoresis Unit, 220V EPS 601 Power Supply 18-1130-02
DALTsix Gel Caster MultiTemp™ III, Thermostatic Circulator, 115 V
MultiTemp III, Thermostatic Circulator, 230 V Code no.
1
80-6466-46
1
1
1
1
1
1
1
1
80-6466-27
80-6467-22
80-6485-08
80-6485-27
18-1130-02
80-6485-46
18-1102-77
18-1102-78
Image acquisition
Ettan DIGE Imager
Typhoon Trio™ with ImageQuant™ TL and PC Typhoon Trio+ with ImageQuant TL and PC Typhoon 9400 with ImageQuant TL and PC Typhoon 9410 with ImageQuant TL and PC 63-0056-42
63-0055-88
63-0055-90
63-0055-79
63-0055-81
Image analysis
DeCyder 2-D v7.0 Software Package
DeCyder 2-D v7.0 Oracle 10gR2, 5 user license
1
DeCyder 2-D v7.0, 1 user license
1
28-9435-83
28-9435-88
28-9442-75
Related literature
Code no.
2-D Electrophoresis: Principles and Methods
Ettan DIGE System User Manual
80-6429-60
18-1173-17
GE, imagination at work, and GE monogram are trademarks of General Electric Company.
Cy, CyDye, DeCyder, Ettan, ImageQuant, Immobiline,
IPGphor, Multiphor, MultiTemp, Typhoon, and Typhoon
Trio are trademarks of GE Healthcare companies.
© 2005–2009 General Electric Company —
All rights reserved. First published Nov. 2005
GE Healthcare UK Limited
Amersham Place
Little Chalfont
Buckinghamshire, HP7 9NA
UK
GE Healthcare Bio-Sciences Corp.
800 Centennial Avenue, P.O. Box 1327
Piscataway, NJ 08855-1327
USA
GE Healthcare Europe GmbH
Munzinger Strasse 5, D-79111 Freiburg
Germany
GE Healthcare Bio-Sciences KK
Sanken Bldg., 3-25-1, Hyakunincho
Shinjuku-ku, Tokyo, 169-0073
Japan
imagination at work
11-0013-40 AD 01/2009
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