Biology Reference
In-Depth Information
4. Porcine modifi ed trypsin (Promega, Madison, WI, USA).
Create a stock solution by resuspending the lyophilized mate-
rial at 100 ng/mL in 5 mM NH
4
HCO
3
and store in 10 mL
aliquots at −80°C. The 10 ng/mL working solution is made
with the addition of 90 mL of 5 mM NH
4
HCO
3
to the 10 mL
aliquot, which should be thawed and maintained on ice. This
is done immediately before use.
5. Acetonitrile.
6. Formic acid.
3. Methods
1. Dilute 20 mL of mouse serum in 90 mL of MARS buffer A.
Add to the concentrator pocked of the 0.22-mm centrifugal
fi ltration device and spin for 1 min at 16,000 ×
g
.
2. Program the HPLC system with the following method:
0-10 min: 100% MARS buffer A at 0.5 mL/min; 10.01-17 min:
100% MARS buffer B at 1 mL/min; and 17.01-28 min: 100%
MARS buffer A at 1 mL/min. The maximum operating
pressure is 120 bar.
3. Prime the HPLC system (see Note 2).
4. Inject 100 mL of the diluted serum at 0.5 mL/min. Collect the
“fl ow-through” peak, which contains the low-abundance pro-
teins, into a 15-mL Falcon polypropylene tube. This elutes
from the column between 1.5 and 5.5 min after commencing
the run and has a volume of ~2 mL. Immediately, add 8 mL of
ice-cold acetone to the fl ow-through peak and store on ice
while the “bound” peak is collected.
5. Collect the bound peak containing the high-abundance pro-
teins (albumin, transferrin, and IgG for the MARS-M3 col-
umn) into a 50-mL Falcon polypropylene tube. This elutes
between 12 and 16 min following the commencement of the
run and has a volume of ~4 mL. Add 16 mL of ice-cold acetone
to the bound fraction, then immediately store both fractions at
−20°C for at least 16 h to allow proteins to precipitate. The
samples should be processed within 5 days of their collection
or transferred to −80°C.
3.1. Depletion
of High-Abundance
Proteins
1. Recover the protein pellets by centrifugation at 5,000 ×
g
for
10 min at 10°C. Remove the supernatants, ensuring the pro-
tein pellets remain intact. Invert the tubes onto a lint-free
paper towel (e.g., Kimwipe, Kimberly-Clark, Dallas, TX, USA)
for 5 min to ensure maximum liquid removal and transfer into
a fresh tube.
3.2. Sample
Preparation for DIGE
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