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Difference
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References: Tonge,
R, Shaw, J, Middleton, B, Rowlinson, R, Rayner, S, Young, J, Pognan, F,
Hawkins, E, Currie, I, and Davison, M. 2001. Validation and development of
fluorescence two-dimensional differential gel electrophoresis proteomics
technology. Proteomics,
1:377-396 DiGE
Protocol: 1.
Please see the Ettan DIGE User Manual
for all buffer recipes and for more detailed directions - the following provides
only an overview. 2.
Resuspend cell pellets in 1 ml Standard Cell Wash Buffer
in a 1.5 ml microcentrifuge tube. Note: 1 x 106 tissue culture
cells or 3 x 108 bacterial cells contain approximately 50 礸 protein. 3.
Pellet the cells (12,000 g for 4 min in a refrigerated
bench top microfuge) and repeat steps 2 and 3 at least once more to remove
all media which in the case of cell culture media may contain large amounts
of albumin. 4.
Lyse the cells with a volume of Lysis Buffer (7M urea,
2M thiourea, 4% CHAPS, in 25mM tris, pH 8.6, @ 40C) such that the
final protein concentration should be from 5-10 mg/ml 5.
Centrifuge the cell lysates and confirm with pH paper
that the pH is still at 8.6. 6.
Store cell lysates in aliquots at -70 degrees C while
protein assays are carried out on aliquots of all lysates. 7.
Use hydrolysis/amino acid analysis (available through
the Keck Laboratory) or the PlusOne 2-D Quant Kit (GE Healthcare), which is
compatible with detergents, to determine the protein concentrations in all
samples. 8.
In vitro label 50ug of the control protein extract and
50ug of the experimental protein extract with GE Healthcare Cy-3 and Cy-5
N-hydroxysuccinimidyl ester dyes. These dyes have been matched with respect
to charge and mass - with the single positive charge of the dye replacing the
charge lost by the modified lysine or N-terminus of the protein. Cy-3 and
Cy-5 labeled proteins co-migrate - with the dye label adding approximately
450 Da to the proteins in each sample. 9.
Optional (highly recommended) use of a third dye (Cy-2)
as an internal (pooled 25ug control + 25ug experimental) standard to permit
normalization of multiple gels and for internal normalization. 10. Mix control,
experimental, and internal standard samples together (i.e., 150ug total
protein) and then add an equal volume of 2X Sample Buffer. 11. Bring volume to 450
ul with Rehydration Buffer, rehydrate 24 cm Immobiline (IPG) Drystrips (GE
Healthcare) for 10-24 hrs, and carry out isoelectric focussing. Currently,
the Keck Laboratory is using pH 3-10 IPG strips unless requested otherwise.
Note that other pH ranges that are available include: 3-7, 4-7, 3.5-4.5,
4.0-5.0, 4.5-5.5, 5.0-6.0, 5.5-6.7, and 6-9. 12. If necessary, after
isoelectric focusing the IPG strip may be stored in an equilibration tube (GE
Healthcare) at -70 degrees C for at least several months. 13. Carry out the SDS
polyacrylamide gel electrophoresis (second) dimension on a 10 inch wide by
7.5 inch tall by 1.0mm thick gel with one side coated with Gelbond.
Currently, the Keck Laboratory routinely uses a 12.5% polyacrylamide gel
which will optimally separate 12-100 kD proteins. 14. Immediately after
SDS PAGE, the gel (which is still held between two glass plates) is scanned
at all 3 wavelengths simultaneously on a GE Healthcare Typhoon 9410 Imager.
After scanning, 16 bit tiff files of each color channel are exported for
image analysis using the differential in-gel analysis module of the GE
Healthcare DeCyder software package. After spot detection (which includes
automatic background correction, spot volume normalization and volume ratio
calculation), a user defined "dust filter" may be applied to each
gel. This has the effect of automatically removing non-protein spot features
from the gel and is followed by recalculation of experimental parameters. 15. The front glass
plate is removed and the gel is then fixed and stained with Sypro Ruby, which
is the fluorescent stain that will be used as a guide to excise spots of
interest from the gel. The reason for using Spyro Ruby, which stains all
protein in the gel, is that the Cy-dye labeling is carried out such that the
extent of incorporation will be <5% in terms of mole Cy-dye/mole protein.
Since the Cy-dye has a MW of about 580 Da, low MW proteins (e.g., 10 Kd)
labeled with Cy-dyes will not exactly co-migrate in the SDS PAGE dimension
with their non-labeled counterparts. 16. GE Healthcare
DeCyder software is used to quantify the gel image and to identify a
"pick list" of differentially expressed protein spots to be excised
and subjected to MS-based protein identification. The DeCyder software can
analyze any two Cy-dyed gel images, either on the same gel or on different
gels, match the spots between the two images, and then identify
differentially expressed protein spots. The DeCyder software automatically
outputs a listing of statistically significant differences in protein
expression including t-test values, using the Cy-2 internal standard. Please
note, however, that replicate samples are required for statistical analysis.
Differentially expressed spots may be identified using a number of criteria
including area, volume, 3D peak slope, 3D peak height, and/or statistical
variation. Protein spots that show different degrees of intensity between the
two samples will be highlighted by the software so they can be manually
confirmed. The DeCyder software can also analyze Sypro Ruby images, match the
spots found with Sypro staining to those identified with the Cy-dye stains,
and then choose a ick list?from the Sypro stained gel image. DeCyder data can
be read by labs without the DeCyder software using an HTML format. 17. The protein spot
pick list is transferred to the Ettan Spot Picker instrument (GE Healthcare)
which automatically excises the selected protein spots from the gel and
transfers them into a 96-well microtiter plate. 18. The excised protein
spots are then subjected to automated in-gel tryptic digestion on the Ettan
TA Digester. 19. An aliquot of each
digest is spotted (along with matrix) onto a MALDI-MS target. 20. High mass accuracy,
automated MALDI-MS/MS spectra are acquired on each target (using the Keck
Laboratory抯 Applied Biosystems
4800 Tof/Tof instrument) and the resulting peptide masses are subjected to
database searching using Mascot algorithms. 21. The remaining
aliquots of digests of protein spots that are not identified by this approach
may be subjected to nanospray or LC/MS/MS analysis (Micromass Q-Tof) with the
resulting MS/MS spectra then being subjected to Sequest database searches to
identify proteins present in the sample. 22. Amersham Biosciences
DIGE Instruction Manual |