Biology Reference
In-Depth Information
15. Add CBB staining solution to completely cover the gels. Stain
overnight, slightly agitating.
16. Remove the CBB staining solution and destain four times for
30 min with water.
3.3 In-Gel Digestion
Work cleanly and cautiously to avoid keratin contamination of the
samples. Use an agitator for Eppendorf tubes, which allows rate
300-600 RPM and setting temperature.
1. Excise the bands of interest from the gel using a scalpel. We
were able to distinguish six OB proteins in the profi le (Fig.
1
).
Cut the protein bands into small pieces with an edge of 1 mm
(
see
Note 16
).
2. Collect the gel pieces from corresponding bands in all four
lanes into one Eppendorf tube. Usually, 70
μ
L of solution is
enough to cover gel pieces from four lanes.
3. Wash the gel pieces with 70
μ
L of water for 5 min at room
temperature.
4. Remove water and add around 70
L of washing buffer/ACN
mixture to destain the gel. Agitate at room temperature for
20 min. Repeat twice or three times to completely destain the
gel pieces.
5. Remove the solution and add 70
μ
L of ACN and keep in for
1 min to dehydrate the gel pieces. Repeat, if needed, to com-
pletely dry the gel.
6. Add 70
μ
L of DTT solution and allow the dry pieces to soak.
Make sure that all the pieces are covered with the solution.
Agitate at 56 °C for 45 min.
7. Cool the tubes to room temperature, remove the DTT solu-
tion, and add 70
μ
L of IAA. Agitate at room temperature in
the darkness for 30 min.
8. Wash the gel pieces with washing buffer/ACN mixture for
15 min at room temperature.
9. Cool the agitator to 4 °C (
see
Note 17
).
10. Remove the solution from tubes, add 70
μ
L of ACN, and keep
in for 1 min to dehydrate the gel. Repeat, if needed, to com-
pletely dry the gel pieces.
11. Prepare protease solution, trypsin, chymotrypsin, or their mix-
ture. Add 30-60
μ
L of protease solution to the gel pieces and
allow them to soak at 4 °C. Make sure that all the pieces are
covered with the solution. Agitate at 4 °C for 45 min.
12. Remove the rest of protease solution and add 30-60
μ
L of
50 mM ammonium buffer to completely cover the gel
pieces.
μ
