Biology Reference
In-Depth Information
tank. Fill the top reservoir with SDS-PAGE running buffer.
Check for leaks from the top into the bottom compartment.
Rinse out any non-polymerized acrylamide solution from the
wells using electrophoresis buffer.
5. Apply the sample by using a syringe and add carefully up to
30
L of sample to the bottom of each well. The volume and
protein concentration of the sample should be suffi cient to
give at least 10
μ
L of the molecular
weight standards to one or two wells, preferably in an asym-
metric position.
6. To perform the electrophoresis running, close the apparatus
with the lid and connect the wires to the power supply unit
(see
Note 20
). Apply 200 V for ~45 min in this system. Run
the gel until the blue dye front reaches the bottom of the gel
(
see
Note 21
). Disconnect the electrophoresis unit from the
power supply, remove the lid, and discard the SDS-PAGE running
buffer. Separate the gel from plates with the help of a spatula,
discard the stacking gel, and wash the separating gel with distilled
water to remove traces of SDS-PAGE running buffer.
μ
g of each protein. Apply 15
μ
3.5
Gel Staining
The selection of Coomassie blue or silver gel staining is dependent
on the quantity of protein that samples contain. Silver gel staining
is usually used when samples contain nanograms of proteins.
All steps should be performed with gentle shaking of the staining
tray at room temperature.
1. For silver gel staining (
see
Note 22
), gels are stained according
to the method of Blum et al. 1987 [
32
]. Fix gel for at least
30 min in fi xative solution. Transfer gel to incubation solution
1 for 15 min. Decant carefully the fi xer and rinse the gel three
times for 5 min with deionized water to remove the fi xative
solution. Transfer gel to incubation solution 2 for 2 min.
Decant thiosulfate carefully and rinse the gel three times for
30 s with deionized water to remove the incubation solution.
Transfer gel to incubation solution 3 for 25 min. Decant silver
nitrate carefully and rinse the gel three times for 1 min with
deionized water to remove the incubation solution. Transfer
gel to developing solution and leave for a maximum of 10 min.
Transfer gel to the stop staining solution for 10 min. Rinse
repeatedly gel with water to remove residual stain.
For silver gel destaining, cut the interest bands from
gels and destain with destaining solution until no bands are
visible. The gel will have a yellow hue. Rinse gel repeatedly
(fi ve times, 5 min) with water until gel is transparent and has
no background color (Fig.
1
). Place bands in different tubes
containing distilled water until their processing.
