Biology Reference
In-Depth Information
5. Ice-cold 100 % (v/v) trichloroacetic acid (TCA) was added to
the supernatant in a fresh tube to a fi nal concentration of 25 %
(v/v). The solution was vortexed briefl y, and proteins were
precipitated overnight at −20 °C.
6. The protein suspension was centrifuged at 17,000 ×
g
for
10 min at 4 °C to pellet the precipitate. The supernatant was
removed and the protein pellet was washed twice with 850
μ
L
ice-cold acetone.
7. The protein pellet was air dried for 5 min until the acetone
evaporated.
8. The protein was solubilized in 100
L of 2× SDS sample buffer,
without DTT or bromophenol blue, and a BCA assay was per-
formed to determine the concentration of the protein sample
using bovine serum albumin (BSA) as a standard (
see
Note 3
).
9. 1 M stock solution of DTT was added to 100
μ
g of protein
extract to give a fi nal concentration of 40 mM DTT. A trace of
bromophenol blue was added, and the mixture was heated at
75 °C for 5 min before fractionation by SDS-PAGE using a
10 % precast gel and 1× SDS running buffer. Proteins were
electrophoresed at 70 V for 15 min, followed by 160 V for
50 min.
10. The gel was lightly stained with Coomassie blue to provide a
visual aid for subsequent handling, although this is not strictly
necessary.
μ
3.2 In-Gel Digestion
Procedure
This procedure was originally adapted from Shevchenko et al. [
25
].
1. The stained gel was placed on a clean glass plate, and the lane
containing the protein extract was excised from the gel using a
clean scalpel (
see
Note 4
). The lane was then cut into 16 equal-
size fractions from top to bottom of the gel, and each of the 16
gel bands was cut into smaller fractions (
see
Note 5
) (Fig.
2
).
2. The 16 groups of gel pieces were transferred to 0.65 mL poly-
propylene tubes or 96-well plate and excess water removed.
The gel pieces were washed briefl y with 100 mM NH
4
HCO
3
to ensure correct pH of gel pieces.
3. The gel pieces were destained with 200
L 50 % (v/v) aceto-
nitrile (ACN) and 50 % (v/v) 50 mM NH
4
HCO
3
by vortex-
ing, and then incubating for 10 min at room temperature. The
liquid was removed, and then this step was repeated. The gel
pieces should be clear and free of stain at this stage. If the gel
pieces are still stained, then repeat the above washing as neces-
sary. However, it is allowable to proceed with traces of stain, as
it will be removed during further washing steps (
see
Note 6
).
4. The gel pieces were washed for 5 min with 200
μ
L 50 % (v/v)
ACN and 50 % (v/v) 50 mM NH
4
HCO
3
, and then 5 min with
μ
