Biology Reference
In-Depth Information
8. Centrifuge at 25 °C for 20 min at 16.000 ×
g
.
9. Transfer supernatant to a new tube.
10. Determine protein concentration using the Bradford or the
BCA assay (
see
Note 3
).
11. Prior to loading to the SDS-PAGE gel, add DTT to a final
concentration of 20 mM and (optionally) bromophenol blue
to a final concentration of 0.01 %.
12. For storage, freeze samples in liquid nitrogen and store at
−80 °C.
3.2 SDS-PAGE
(See Note 7)
1. Assemble gel apparatus, and fill with 1× Tris-Glycine (Laemmli)
SDS-PAGE running buffer.
2. Carefully remove comb and rinse wells with 1× Tris-Glycine
(Laemmli) SDS-PAGE running buffer using a loading tip or
syringe.
3. Load protein sample yielding 200-400 μg protein per well and
molecular weight marker.
4. Run the gel at constant current (25 mA/gel).
5. Stain the gel with Coomassie brilliant blue.
1. Place gel on a clean glass plate.
2. Cut each lane of the gel into 15-20 sections.
3. Excise bands with a clean scalpel and transfer to a 1.5 ml
Eppendorf Safe Lock tube.
3.3 Cutting the Gel
(See Note 8)
1. Add 100 μl H
2
O to the excised gel slices. Shake for 10 min at
room temperature. Take off and discard the supernatant.
Repeat this step once.
2. Add 100 μl destaining solution. Shake for 15 min at room
temperature. Take off and discard the supernatant. Repeat this
step four times.
3. Add 100 μl H
2
O. Shake for 15 min at room temperature. Take
off and discard the supernatant. Repeat this step once.
4. Add 100 μl ACN. Shake for 15 min at room temperature. Take
off and discard the supernatant.
5. Dry the gel slices in a vacuum concentrator.
3.4 In-Gel Digest
(See Note 9)
3.4.1
Destaining
1. Add 40 μl reducing solution. Shake for 5 min at room
temperature.
2. Incubate for 30 min at 50 °C. Take off and discard the
supernatant.
3. Add 100 μl ACN. Shake for 15 min at room temperature. Take
off and discard the supernatant.
3.4.2 Reduction and
Alkylation of SH-Groups
