Biology Reference
In-Depth Information
and balance them in pairs. Ultracentrifuge (w/w) at
231,000 ×
g
for 35 min (
see
Note 18
).
7. Discard the supernatant. Add an appropriate quantity of
PM-suspension medium to each tube and homogenize the
pellets with a Tefl on-glass homogenizer. After balancing in
pairs, ultracentrifuge at 231,000 ×
g
for 35 min.
8. Discard the supernatant with an aspirator. Add a minimal
quantity of PM-suspension medium to the sample tube. Break
up the pellets with a glass rod, transfer into a Tefl on-glass
homogenizer, and homogenize well using an electric Tefl on-
glass homogenizer (moving up and down fi ve times). Transfer
into a 1.5 mL microtube. The DRM fraction should be frozen
in liquid nitrogen immediately and stored at −80 °C.
3.3 In-Gel Tryptic
Digestion
1. Mix 2.5 mL running gel solution, 3.35 mL 30 % (w/v) acryl-
amide solution, and 3.95 mL water in a conical fl ask. Degas
with a vacuum pump for 5 min. Add 100
μ
L of 10 % (w/v)
3.3.1 SDS
Polyacrylamide Gel
Electrophoresis
L
of TEMED. Cast gel into a 90 mm (W) × 83 mm (H) × 1 mm
(T) gel cassette immediately. Insert a 14-well comb without
introducing air bubbles. Incubate at room temperature for 1 h
(
see
Note 19
).
2. Mix 5
ammonium persulfate, 100
μ
L of 10 % (w/v) SDS, and 5
μ
L) and
equal volume of SDS sample buffer. Vortex and centrifuge
tubes briefl y. Heat at 95 °C for 5 min. Centrifuge and cool to
room temperature.
3. Wash out the wells by pipetting up and down. Slowly load
the samples onto the gel. Electrophorese at 100 V until
the upper end of sample dye band enters 2 mm from the well
(
see
Note 20
).
4. Pry the gel plates open with a knife. Cut out the gel slice from
the well to 2 mm in front of the BPB dye with a scalpel on a
glass plate. Cut the gel slice into four equal pieces (Fig.
2
) and
put into 1.5 mL microtubes (
see
Note 21
).
5. Add 200
μ
g of PM or DRM protein samples (within 10
μ
L of fi xation solution and agitate for 10 min.
Centrifuge briefl y and discard the supernatant. Repeat these
steps twice.
μ
All of these procedures must be performed at room temperature
unless otherwise specifi ed.
3.3.2 In-Gel Tryptic
Digestion for Nano-LC-
MS/MS
1. Add 200
L of water and agitate for 10 min. Centrifuge briefl y
and discard the supernatant.
2. Add 400
μ
L of 25 mM ammonium bicarbonate/50 % (v/v)
acetonitrile and agitate for 10 min. Centrifuge briefl y and dis-
card the supernatant.
μ
