SDS-PAGE

[Alex Chang]

Hi, everyone:

Something wired happened to my SDS-PAGE gel. I did SDS-PAGE many times
before, but haven't done it for a while. Recently, when I adapted my old
protocal for a mini-gel, it takes hours for the separating gel to
solidify and even worse, the stacking gel solidified poorly and the wells
did not contain the samples very well as the result of it.

The way I prepare the separating gel is that I don't deareate it. After
I pour a gel into the assembly I add in some isobutanol right away. I
found after the gel solidified, there is some water in between the
isobutanol and the gel.

It takes less time now (1 hr) for the separating gel to solidify after I
increased TEMED from 5 ul/10ml to 20 ul/10ml (10% Ammonium persulfate
unchanged: 50 ul/10ml).

So, my biggest problem is the water formation after solidification of the
separating gel and the problem with the stacking gel. The recipe I am
using is attached. I am appreciating very much if someone could give me
some idea to improve my assay.

Separating gel:
30% Acrylamide:Bis (30%T, 2.67% C)      4.17 ml (12.5%)
dH2O                                    3.2 ml
1.5M Tris-HCl (pH 8.8)                  2.5 ml
10% SDS                                 0.1 ml
10% Ammonium persulfate                 50 ul
TEMED                                   20 ul
Total                                   10 ml

Stacking gel:
30% Acrylamide:Bis              0.67 ml (4%)
dH2O                            3 ml
0.5 M Tric-HCl (pH 6.8)         1.25 ml
10% SDS                         50 ul
10% Ammonium persulfate         25 ul
TEMED                           10 ul
Total                           ~5 ml


Alex

Alex Chang
Pathology
University of British Columbia
achang at hivnet.ubc.ca