It is not the lack of polymerization that this "myth" is talking about. Many years ago, towards optimising semi-quantitative conditions for westerns, I did a detailed study on all the variables that affect polymerization of polyacrylamide including efect of tris, sds, polyerization temperatures, TEMED from several companies and APS (stored or freshly prepared) as well as transfer conditions for westerns. The lack of polymerization is usually due to very bad acrylamide rather than APS or TEMED. Even bad APS, old 4C stored aliquots (which are not ideal storage conditions for APS, since they tend to get oxidised on storage) or old TEMED still had sufficient undegraded molecules to behave as proton donors and catalysts towards polymerisation. But using FRESH APS (or -20C frozen aliquots as well) or properly stored TEMED, as well as the correct molarity of Tris (freshly made within 1 month) or SDS (no more than a month old) had significant (statistically) improvement in band ! resolution (I used a Cytochrome C band which is doublet under good resolution) and uniformity in polymerization. The degradative products primarily of acrylamide and then APS do affect polymerization, though people satisfied with suboptimal polymerization may never notice the difference. I observed that very few publications could ever get the doublet for cytochrome C, which I was able to obtain time and agian consistently with my conditions. TO say the truth, my studies just proved what >3 decades of SDS-PAGE techniques have shown.. Use FRESH reagents whenever possible. I hope this rests the controversy. Jay
________________________________________ From: [email protected] [[email protected]] On Behalf Of Michael Sullivan [[email protected]] Sent: Monday, October 24, 2011 12:36 PM Cc: [email protected] Subject: Re: Hello...Native Page is not polymerising --- On Oct 24, 2011, at 10:02 AM, AllisonH wrote: > On 24/10/2011 3:54 AM, DK wrote: >> In article<[email protected]>, >> lautys<[email protected]> wrote: >>> APS should prepare fresh every time you do your gel. >> >> For how much longer will this myth be propagated? >> >> There is nothing wrong with aliquoting APS and storing it at -20. I've used >> APS >> solutions that were certain to be close to a year old stored at -20C and it >> still >> worked fine with no obvious change in polymerization time (0.05-0.1% APS >> final). >> >> DK > > I was going to jump in with a similar comment but you beat me to it! > I also aliquot my APS and store at -20C with no loss in activity. It's also > ok for a several days at 4C (I used it up to a week later). Sure beats > having to weigh it out each time. > Allison > In fact I've used APS solution stored at 4 degrees for more than a month with no polymerization problems. _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods This email message may contain legally privileged and/or confidential information. If you are not the intended recipient(s), or the employee or agent responsible for the delivery of this message to the intended recipient(s), you are hereby notified that any disclosure, copying, distribution, or use of this email message is prohibited. If you have received this message in error, please notify the sender immediately by e-mail and delete this email message from your computer. Thank you. _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
