-
Please see the
Ettan
DIGE User Manual for all buffer recipes and for more detailed
directions - the following provides only an overview.
-
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 µg protein.
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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.
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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
-
Centrifuge the cell lysates and confirm with pH
paper that the pH is still at 8.6.
-
Store cell lysates in aliquots at -70 degrees C
while protein assays are carried out on aliquots of all lysates.
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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.
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In vitro label 50 µg of the control protein
extract and 50 µg 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.
-
Optional (highly recommended) use of a third dye
(Cy-2) as an internal (pooled 25 µg control + 25 µg experimental) standard to
permit normalization of multiple gels and for internal normalization.
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Mix control, experimental, and internal standard
samples together (i.e., 150 µg total protein) and then add an equal volume of 2X
Sample Buffer.
-
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.
-
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.
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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.
-
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.
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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.
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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 ‘pick list’ from the Sypro stained gel image. DeCyder
data can be read by labs without the DeCyder software using an HTML format.
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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.
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The excised protein spots are then subjected to
automated in-gel tryptic digestion on the Ettan TA Digester.
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An aliquot of each digest is spotted
(along with matrix) onto a MALDI-MS target.
-
High mass accuracy, automated MALDI-MS/MS spectra are
acquired on each target (using the Keck Laboratory’s Applied Biosystems 4800 Tof/Tof
instrument) and the resulting peptide masses are subjected to
database searching using Mascot algorithms.
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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.
