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- SEQUEST software.
- DTASelect software.
- Custom Perl scripts: coverage.pl, protomap.pl, and peptogra-
pher.pl (available at http://www.scripps.edu/chemphys/
cravatt/protomap ).
3 Methods
- Prepare 200 μg of each protein sample in 4× SDS-PAGE loading
buffer ( see Note 1 ). - Load sample fl anked by prestained molecular weight markers
onto a 10 % acrylamide gel ( see Note 2 ). - Run the gel at constant 250 V/h until there are approximately
11 cm between the 250 kDa MW marker and the dye front. - Remove the gel from the plates. Cut off the stacking gel and
any excess gel below the dye front ( see Note 3 ). - Place the gel in a square glass dish and rinse in high-purity
water for 10 min. - Clean a glass plate with water and then rinse with methanol
and dry completely. - Place a paper grid with lines spaced 0.5 cm apart underneath
the glass plate, and place the gel on top of the glass. - Line up the top molecular weight marker (250 kDa) in the fi rst
lane as well as the last lane on top of one of the lines. - Using a razor, cut one band above this marker and then cut
across all gel lanes horizontally every 0.5 cm following the
guide lines ( see Note 4 ). - As soon as the gel is cut horizontally, cut the gel vertically in
the middle of the MW markers to separate distinct samples. - One by one, take a single slice of gel and cut into small pieces
(approx 1 mm^2 ) and place into microcentrifuge tubes contain-
ing 500 μL 100 mM ammonium bicarbonate. - Repeat for every gel slice for every sample (usually 22 bands
per sample), placing each in a separate microcentrifuge tube
with 500 μL 100 mM ammonium bicarbonate.
Note: All washes are performed with slow vortexing.- Wash each gel band 2× for 15 min with 500 μL 100 mM
ammonium bicarbonate. - After wash(es), remove supernatant and add enough 10 mM
TCEP to completely cover gel band (approx 200 μL). - Place at 37 °C for 30 min.
- Quick spin and remove TCEP with aspirator or pipetman.
2.4 Data Analysis
and Generation of
Peptographs
3.1 10 % SDS-PAGE
and Cutting the Gel
3.2 In-Gel Digestion
( See Note 3 )
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