Biology Reference
In-Depth Information
Well
2 mm
2 mm
Cut out
Cut into
4 pieces
Put into
microtube
Fig.
2
Excision of a protein band from gel. Wells are separated to avoid contami-
nation of different samples during loading and electrophoresis. After the BPB dye
migrates into a gel (about 2 mm), a 4 mm of gel piece centered on the BPB dye
band is cut out. Subsequently, the gel piece is cut into four equal pieces. Each of
the gel pieces is separately put into 1.5 mL microtubes
3. Add 200
L of acetonitrile and incubate at room temperature
for 5 min. Centrifuge briefl y and discard the supernatant
(
see
Note 22
).
4. Add 100
μ
L of 0.1 M ammonium bicarbonate and centrifuge
briefl y. Incubate at room temperature for 5 min (
see
Note 23
).
5. 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
).
6. Dry out the gel samples using a centrifugal concentrator for
45 min (
see
Note 25
).
7. Add 100
μ
L of reduction buffer and centrifuge briefl y.
Incubate at 56 °C for 45 min. Discard the supernatant.
8. Add 100
μ
L of 55 mM IAA/0.1 M ammonium bicarbonate
and centrifuge briefl y. Incubate in the dark at room tempera-
ture for 30 min. Discard the supernatant.
9. Add 200
μ
L of water and agitate for 10 min. Centrifuge briefl y
and discard the supernatant.
μ
