lpss at unixg.ubc.ca (Alex Chang) wrote:
>>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.
>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
>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
>University of British Columbia
>achang at hivnet.ubc.ca
It might help if you change the ratio of TEMED to APS a little bit. I use (for 10 ml) 35 micro-l 10% APS and 14 micro-l TEMED. After 30 min you can pour the stacking gel, but you should wait at least 1 h before you use the gel. For best separations I pour the separation gel the evening before and the stacking gel the next day (when I use it).
Another problem might be (I) your TEMED is to old (II) your acrylamide is too old (III) your plates are not clean (maybe you or your college applied vaccum grease to the plates to seal a leaking apparatus. If so, you should place your plates o/n in 1-2 N NaOH and wash them afterwards as usual).
Usually the problem is solved with fresh TEMED or fresh acrylamide.
BTW, I don't use the Laemmli system any more and use the system by Fling and Gregerson instead because of the much better separation (Anal. Biochem. 155: 83-88 (1986).
Hope that helps
Institute for Molecular and Cellular Biology
16 Divinity Ave.
Cambridge, MA 02138
Tel: (USA) 617 495 3716; Fax: (USA) 617 495 9300
Email: Thomas at hastingslab.harvard.edu