Acrylamide polymerisation with TeMed

BradAmos

I have not tried the CLARITY method yet, but some have reported difficulties with polymerisation. I noticed when preparing polyacrylamide gels for electrophoresis that plastics, e.g. vinyl tubing give off a powerful inhibitor of polymerisation.

joelrosiene

TeMed doesn't seem to work. It works for your normal acrylamide gels, but not for the Clarity gel ratio. You have to order the special VA-044 initiator that they referenced in the paper.

liz_kritzer

I initially had trouble polymerizing my samples, but can confirm that it does actually work as described in the protocol. I have successfully polymerized samples in a variety of polypropylene vessels, but I don't know about other types of plastic.

I think the other problem with TeMed would be that all the components of the hydrogel have to seep into the tissue. Since polymerization happens pretty quickly upon the addition of TeMed, I'm not sure if it would have time to diffuse into all parts of your tissue.

KatherineHolzem

@BradAmos... are you per chance trying to use homemade 16% PFA. It doesn't quite make sense to me, but I have made several batches of hydrogel solution at this point, and the only batch that didn't polymerize (or, I should say, just polymerized very, very slowly) was the batch with homemade PFA. Every time I have used the pre-made stuff from EMS, it's worked.

I agree with @liz_kritzer that TeMed is probably not the best because you cannot control the cross-linking initiation. According to one of my labmates who has experience with hydrogels, VA-044 is not the only initiator that would work for this reaction... It's just super cheap and effective.

willadler

I just want to note that we use our own homemade 16% Fisher brand PFA, and it polymerizes fine.

liz_kritzer

I've had mixed results with homemade PFA. Some batches polymerize fine, and some fail entirely. I can't identify any difference in preparation between batches, but have started using the pre-made 16% from EMS since it seems more reliable.

rchurt

@willadler, what was the pH of your solution?

It looks like, especially with TeMed, pH is important, and the formaldehyde solution is the component that will contribute the most to the pH of your hydrogel solution if you're using a base to catalyze the depolymerization of PFA when you make it. Check this out: http://onlinelibrary.wiley.com/doi/10.1002/elps.1150140184/abstract