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1.4 Image Analysis
Image analysis is extracting tangible data out of
the 2DE image with specially designed software.
Typically it involves detecting spots and warping
separate images to align like-spots of the same
protein. Spot data comes from the level of spot
darkness which is proportional to the level of
protein staining or dye labeling of particular amino
acids.
3D-views of differentially expressing protein spots
Image analysis is a powerful technique which can
readily identify differences between gel images.
Similarities between gel images can also be
elucidated but most queries biologists have concern
differential protein expression. Image analysis
software can group your samples into separate
biological groups such as Control and Diseased and
then identify proteins for biomarker candidacy.
Creative Proteomics can analyze traditional single
stain and DIGE experiments.
2. Quantitative and
Comparative Proteomics Using 2D DIGE
(Two-Dimensional Difference Gel Electrophoresis)
Services
2.1 Features
· This
procedure can be multiplexed using any number of
samples compared across gels(using an internal
standard) in a multiplexed 2D gel experiment.
-
Typically, differential expression ratios are
obtained on 1,000 or more protein spots.
-
DIGE provides a top-down view into changes in
the post translational proteome.
-
Protein spots are automatically selected via
user-defined criterion (e.g., differential
expression between a control versus cancer
sample of >1.3-fold) and subjected to robotic
spot excision, trypsin digestion, and
MALDI-TOF/TOF (AB Model 4800)
-
Protein identification is based on highly
accurate masses and MS/MS sequence data from
multiple tryptic peptides (e.g., 5-10
peptides/protein is common)
-
Very hydrophobic, basic, or very large or small
molecular weight proteins (> 200K or < 5K) may
not migrate into or resolve on the gel. To
circumvent this possible limitation,
complementary protein profiling analyses of
iTRAQ or label free quantitation can be used.
-
Simplified workflow, from sample prep and
separation to data analysis
-
Allows you to analyze differences in low
abundance proteins with unparalleled statistical
confidence
-
Many of our 2-D electrophoresis products improve
data quality from traditional 2-D experiments as
well, and the products can eventually be
integrated into a complete solution to maximally
improve your 2-D results.
2.2 2D DIGE Principle
DIGE utilizes mass- and charge-matched, spectrally
resolvable fluorescent dyes (Cy2, Cy3 and Cy5) to
label up to 3 different protein samples (or 2
samples and 1 pooled internal standard) in vitro
prior to 2-D electrophoresis. Both the control and
the experimental sample are run in a single
polyacrylamide gel but imaged separately (Typhoon
9410) at a discrete wavelength for each respective
dye. Hence, the images are overlaid without any
‘warping’ which substantially raises the confidence
with which protein changes between samples can be
detected and quantified.
After imaging analysis, another gel is prepared and
subjected to pick-up. Several post-staining methods
are available on request to stain this preparative
gel. Staining methods include: Coomassie Brilliant
Blue® staining, silver staining, Sypro® ruby
staining or other post-staining methods. These
staining methods vary in sensitivity and
compatibility for downstream proteolytic digestion
and mass spectrometry.
For pick-up, those differentiated protein spots can
be cut off from the gel by the integrated picker.
Washing and proteolytic digestion are performed
prior to the mass spectrometry analysis. MS analysis
is commonly performed using Peptide Mass
Fingerprinting (PMF). Peptide sequences can also be
further analyzed by MS/MS.
Preparative gels can also be prepared for
traditional 2DE and be subjected to pick-up and
subsequent procedures.
2.3 Workflow
Our quantitative and comparative proteomics using 2D
DIGE (two-dimensional difference gel
electrophoresis) services include:
a) Experimental design
b) Sample preparation to isolate total proteins
(maybe using ToPI-DIGE protein isolation Kit)
c) Fractionation, quantitation and qualification of
proteins if applicable
d) 2D-DIGE analysis (Staining, First dimension
isoelectric focusing, Second dimension gel
electrophoresis, scanning of gels using a
DIGE-enabled digital scanner)
e) Imaging analysis: Biological variation analysis
of gel images to identify differentially expressed
proteins with statistically significant difference.
f) Spots excision
2.4 Image Analysis of DIGE
Images
Image
analysis of DIGE images is simplified as compared to
traditional single stain approaches due to the
co-migration of up to 3 samples on one gel. All 3
samples can have the same spot segmentation (spot
outline) and will not require warping. Having the
same spot segmentation on the 3 channels will also
improve spot volume quantitation.
2D DIGE of wild type (Cy3-labeled) and mutant
(Cy5-labeled)
The right panel shows an overlay of the two images.
If equal amounts of a protein are present in both
samples the spots will appear yellow; if the protein
is only present in the wild-type the spot will
appear green; if the protein is only present in the
mutant it will appear red. For each spot a Cy3:Cy5
ratio can be assigned reflecting the differing
amounts of a given protein within the two samples.
If the 3rd sample is replaced with an internal
standard (a pool of all your samples) then this can
be used to warp images from different physical gels,
the ease of warping will be greatly improved as the
internal standard image is the same "sample". In
this approach, the internal standard is used to pull
all the spot data in the experiment together. The
internal standard also has a second function which
is to increase the reliability of quantitation
between gels. Spot volume of your samples will be
expressed as a ratio to the internal standard then
the ratios can be directly compared to one another.
Often researchers are not prepared for the amount of
variation in spot data, especially between organisms
of the same species and the same biological
population. Also, there can often be 5 orders of
magnitude in spot volume between your smallest and
largest spot, i.e., 1 to 100 000! Depending on the
given spot data and the experimental aims,
replication should be introduced into your
experimental design at the appropriate levels. If
there is a high level of seemingly random variation
between organisms of the same biological population
then the number of samples should be increased.
Similarly if there is a high amount of variation
between gel replicates, replication should be
introduced at the gel level (certain sample types on
particular pH ranges are prone to technical
variation). Also, equal numbers of samples between
your biological groups is desirable. At Creative
Proteomics, technical variation is minimised by our
experience in sample preparation, electrophoresis,
gel pouring, imaging and image analysis. Customers
are welcome to discuss experimental design with us.
Triplicate gels, n=1059 matched spots
Mean r²=0.984
Two Dimensional – Difference In Gel Electrophoresis
(2D-DIGE) is a gel-based approach for comparative
proteomics using fluorescent tags. Distinct
flurescent tags e.g. Cy 3, 5 and 2 are used to
label samples and a universal internal standard
prior to 1st/2nd dimension electrophoresis. The
fluorescent dyes have been designed so that they do
not alter the charge of proteins or impart
significant electrophoretic mobility differences to
identical proteins. An automated software program is
used to detect, quantify and annotate differentially
expressed proteins. 2D-DIGE offers all the
advantages of 2D-PAGE and overcomes the inherent
disadvantage of variation and reproducibility
problem in a 2D-PAGE.
2.5 Application Field of 2DE
or 2D-DIGE
These procedures are powerful technologies for
comparing complex protein mixtures from biological
samples in proteomics research.
2.6 Sample Amount
Recommended sample amount is 50 to 100µg
2.7 References
1. Lilley, K.S., Razzaq, A. & Dupree, P. (2002)
Two-dimensional gel electrophoresis: recent
advances in sample preparation, detection and
quantitation. Current Opinion in Chem. Biol. 6,
46-50.
2. Swatton, J.E., Prabakaran, S., Karp, N.A.
Lilley, K.S. & Bahn S (2004) Protein profiling
of human post-mortem brain using 2-dimensional
fluorescence difference gel electrophoresis (2-D
DIGE). Mol. Psychiatry 9, 128-143.
3. Karp, N.A., Kreil, D.P. & Lilley, K.S. (2004)
Determining a significant change in protein
expression with DeCyderTM during a pair-wise
comparison using two-dimensional difference gel
electrophoresis. Proteomics 4, 1421-1432.
3. Why run 2D-DIGE instead of
2D gels?
Compared to traditional 2-D gel techniques for
protein profiling, DIGE provides a significantly
more efficient, sensitive, and reliable way to
detect proteins whose expression is altered between
control and treated samples.
In 2D-DIGE (two-dimensional difference in gel
electrophoresis), we use fluorescent dyes to label
proteins, can run up to 3 samples in one gel. There
are many advantages to 2D-DIGE over traditional 2D
gels:
a. High sensitivity.
The CyDye fluorescent dyes have a sensitivity of 0.2
ng/spot, as compared to the sensitivity of Coomassie
at 100 ng/spot. This allows us to run smaller
amounts of protein on the 2D-DIGE gels and results
in much better spot resolution than traditional 2D
gels stained by Coomassie. Scanned gels are
publication quality.
b. High accuracy.
The extremely high spot resolution enables accurate
software-aided spot quantitation and protein
expression comparison between samples. Differences
in protein expression as small as 10% can be
detected. And protein isoforms and
post-translational modifications are easily
visualized.
c. Fewer number of gels.
Since the protein expression patterns from 3
different samples are compared in the same gel,
fewer gels are required per project. In addition,
there is no need to run technical replicates as in
standard 2D.
d. Fast turn-around.
Software aided in-gel analysis enables fast
turn-around time, customers receive a data report
within 2 week after the samples are received.
e. Cost effective.
The advantages of 2D-DIGE allow us to competitively
price our services. The price of the 2D-DIGE gel
includes sample preparation (both standard and
customized), protein concentration determination,
CyDye labeling, isoelectric focusing, 2D SDS-PAGE,
gel scanning, image preparation, quantitative in-gel
analysis of up to 3 pair-wise comparisons, data
report and consultation with us.
4. 2D DIGE Sample Preparation
Sample preparation is the most critical step in
obtaining good results from 2D Electrophoresis.
Samples can be obtained from tissues or cultured
cells. The quality of the gel depends on the types
and amounts of detergents, salts, contaminants, and
other interfering material present in the sample.
Proteins in the samples must be fully denatured,
reduced, and solubilized for effective IEF
separation. Samples are rapidly lysed in a strongly
denaturing solution with brief sonication in the ice
container. Protease inhibitors can be added in the
lysis solution to prevent protein degradation.
Proper conditions for lysis vary greatly depending
on the source of the samples, and should be
determined empirically.
A minimum of 25 ug total protein, regardless of the
source of samples, is required for analytical 2D
DIGE. Depending on the abundance of the target
protein, at least 200 ug total protein may be needed
for prep-DIGE gel, spot picking and mass
spectrometric analysis. Please submit enough sample
protein so that we can complete your project
successfully. We can perform the standard sample
prep steps for the customers. For the plasma and
serum sample immuno-depletion, additional charge
will apply. Please provide us with specific
instructions if special handling is required. If you
wish to lyse the samples yourself, please adjust the
final concentration of total protein to 3-20 mg/ml.
The following are the basic guidelines for protein
lysate preparation:
a)
It is essential that lysis buffer is free of both
primary amines and sulfhydryl groups.
b)
Follow the sample preparation protocol described in
GE Healthcare's DIGE section.
Customers may consider the following sample
preparation modifications to achieve optimal
results.
 Laser
Capture Micro-dissection (LCM) capture to separate
tumor cells from normal cells in the same tissue
sample
FACS
or Cell sorting to separate different cells (cell
cycle stage or cell types)
Protein
Complex isolation and sample fractionation
Cell
surface membrane protein isolation
 Mitochondria
isolation, ER, nucleus, plasma, protein
fractionation
Pre-isolating
the specific cells of interest
a) Bacteria.
Bacterial cells are pelleted by centrifugation.
Pellets are washed twice with cell washing buffer
(10 mM Tris HCl, 5 mM magnesium acetate, pH 8.0).
After the final spin, the supernatant must be
removed as completely as possible. Cell pellets are
quickly frozen in liquid nitrogen or dry ice/ethanol
bath. Frozen cell pellets can be shipped to us on
dry ice.
b) Yeast.
Yeast cells are pelleted by centrifugation. Pellets
are washed twice with cell washing buffer (10 mM
Tris-HCl, 5 mM magnesium acetate, pH 8.0). After the
final spin, the supernatant must be removed as
completely as possible. Cell pellets are quickly
frozen in liquid nitrogen or dry ice/ethanol bath.
Frozen cell pellets can be shipped to us on dry ice.
c) Mammalian
cell lines.
For suspension cultures, cells are pelleted by
centrifugation. Pellets are washed twice with PBS or
cell washing buffer (10 mM Tris-HCl, 5 mM magnesium
acetate, pH 8.0). After the final spin, the
supernatant must be removed as completely as
possible. Cell pellets are quickly frozen in liquid
nitrogen or dry ice/ethanol bath. Frozen cell
pellets can be shipped to us on dry ice.
For adherent cultures, the cell monolayer can be
washed twice with PBS or cell washing buffer (10 mM
Tris-HCl, 5 mM magnesium acetate, pH 8.0). After the
second wash, a small volume of cell washing buffer
is added to the plates (1 ml for each 100-mm plate),
and cells are scraped from the plates. The scraped
cells are transferred to eppendorf tubes, and cells
are pelleted by a 1-minute spin at low speed. Remove
the supernatant as completely as possible. Cell
pellets are quickly frozen in liquid nitrogen or dry
ice/ethanol bath. Frozen cell pellets can be shipped
to us on dry ice.
d)
Enriched cell organelles (nuclei, mitochondria,
membrane fraction, etc.).
Following the purification steps, wash the enriched
cell organelles twice with cell washing buffer (10
mM Tris-HCl, 5 mM magnesium acetate, pH 8.0). If
necessary, neutral substances (such as sucrose) may
be included in the washing buffer. After the final
wash, the supernatant must be removed as completely
as possible. Pellets are quickly frozen in liquid
nitrogen or dry ice/ethanol bath. Frozen pellets can
be shipped to us on dry ice.
e) Purified protein complex.
If you use IP in getting protein complex, please do
the IP and the washing the same way you did before.
At the end, please transfer the beads to a 1.5 ml
plastic tube, and wash the beads once with 0.5 ml of
water (this should get rid of the salt which may
interfere with IEF). Please remove water as
completely as possible after the final wash. The
bead pellets can be kept at -20C and sent to us on
dry ice. We will elute protein complex from the
beads using a buffer that contains 2 M thiourea, 7 M
urea, 4% CHAPS and 30 mM This-HCl pH 8.8.
f) Animal tissue.
About 5-10 mg of animal tissue (approximately 2 mm x
2 mm x 2 mm in size) is needed for 2D DIGE analysis
and the mass spectrometric protein analysis. We
suggest that the contaminating blood be removed
using PBS. Tissues can be flash frozen in liquid
nitrogen after harvest, or directly stored at -80C
freezer. Frozen tissues can be shipped to us on dry
ice.
Tissues that are embedded in OCT blocks are fully
compatible with 2D DIGE analysis. Embedded tissues
can be shipped to us on dry ice.
g) Human tissue.
About 5-10 milligrams of human tissue (approximately
2 mm x 2 mm x 2 mm in size) is needed for 2D DIGE
analysis. Removing the contaminating blood by
washing with PBS is recommended. Tissues can be
flash frozen in liquid nitrogen after harvest. The
tissue is then ground to a frozen powder in a mortar
and pestle. Frozen tissue powder can be shipped to
us on dry ice.
Tissues that are embedded in OCT blocks are fully
compatible with 2D DIGE analysis. Embedded tissues
can be shipped to us on dry ice.
h) Laser captured micro-dissected (LCM) cell population.
A minimum of 20,000 laser captured micro-dissected
cells is required for 2D DIGE analysis. After LCM,
adherent cells are eluted from the caps with small
amount (10-15 l per cap) of lysis buffer. Lysates
are collected (and pooled if more than one cap) in
microcentrifuge tubes, frozen in liquid nitrogen or
dry ice/ethanol bath. Frozen lysates can be shipped
to us on dry ice and we will complete the lysis
step.
I) Samples containing biohazard materials.
We do NOT accept samples containing any biohazard
material. If your samples contain any biohazard
materials, please make sure to deactivate them
completely using the appropriate procedure and
please declare them in the order form. In addition,
we do NOT accept any samples containing Level 4
biohazard material even if deactivated. Please refer
to the following link for the rank of biohazard
material:
http://en.wikipedia.org/wiki/Biological_hazard.
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Gel-based Quantitative and Comparative Proteomics Services, 2DE or 2D DIGE
