SDS-PAGE gel won't get solidified - (May/07/2006 )
I have been using SDS-Page gels 12% for running and 5% stacking gel.But now i am getting problem in stacking gel which does not get solidified. I have prepared all reagents fresh but still i am facing same problem. Plz anyone of us has solve for this prbolem.
I use twice more APS and temed in the stacking gel than in the separating gel
you may have contaminated temed, try another bottle.
your aps stock may be bad, try someone else's.
you may need to degas your gel mix (prior to introduction of the aps, of course). dissolved oxygen scavenges the free radicals during the polymerization reaction.
you may not be seeing the lack of polymerization in your running gel because of the higher acrylamide concentration, but it is probably not fully polymerized, as well.
it should be better if u store your APS by avoid the moisture.
cause i think ur problem due to APS quality.
APS should be stored in a dry dehydration chamber or it absorbs moisture and degrades. you can increase the conc. of APS and TEMED as miselle said. give some extra time for your stacking gel to solidify/ or place your assembly in front of a lamp to enhance polymerization.
all the best
I keep 10% APS small aliquots at -20C.
Usullay they are good for long time but important is not to freeze and thaw many times. I never freeze back APS. I have made aliquots with volumes that fit with what we need per gel or per 2 gels.
How much APS and TEMED do you use in your stacking gel?