Biology Reference
In-Depth Information
10. Add 400
L of 25 mM ammonium bicarbonate/50 % (v/v)
acetonitrile and agitate for 10 min. Centrifuge briefl y and dis-
card the supernatant.
11. Add 200
μ
L of acetonitrile and incubate at room temperature
for 5 min. Centrifuge briefl y and discard the supernatant
( see Note 22 ).
12. Add 100
μ
L of 0.1 M ammonium bicarbonate and centrifuge
briefl y. Incubate at room temperature for 5 min ( see Note 23 ).
13. Add 100
μ
L of acetonitrile and centrifuge briefl y. Incubate at
room temperature for 15 min. Centrifuge briefl y and discard
the supernatant ( see Note 24 ).
14. Dry out the gel samples using centrifugal concentrator for
45 min ( see Note 25 ).
15. Cool to room temperature and put on ice. Add 25
μ
L of pro-
tease solution to each tube and centrifuge the tubes briefl y.
Incubate on ice for 45 min ( see Note 23 ).
16. Discard the supernatant and add 100
μ
L of 0.1 M ammonium
bicarbonate. Centrifuge the tubes briefl y and incubate at
37 °C for 20 h.
17. Agitate for 15 min and add 100
μ
L of acetonitrile. Agitate for
15 min and collect the supernatant ( see Note 24 ).
18. Add 5 % (v/v) TFA/50 % (v/v) acetonitrile and agitate for
15 min. Centrifuge the tubes briefl y. Collect the supernatant
in tubes, as described in step 17 ( see Note 24 ).
19. Dry out the collected supernatant using a centrifugal concen-
trator for 1 h. Add 30
μ
μ
L of 0.1 % TFA. Store at −30 °C
( see Note 26 ).
All of these procedures must be performed at room temperature
unless otherwise specifi ed.
3.4 In-Solution
Tryptic Digestion
1. Precipitate 100
g of PM or DRM protein using an ultracen-
trifuge (231,000 × g , 4 °C, 50 min).
2. Add solution B to the centrifuge tubes and homogenize with
a Tefl on-glass homogenizer. Transfer to 1.5 mL microtubes.
3. Solubilize samples and measure the protein concentration
with a Pierce BCA protein assay kit according to the instruc-
tion manual from the manufacturer.
4. Transfer 5
μ
g of PM protein to another 1.5 mL microtube.
Make up to 20
μ
L with solution A.
5. Perform reductive alkylation and tryptic digestion according
to the instruction manual, and store at −30 °C ( see Note 26 ).
μ
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