I'm trying to identify a novel enzyme using a zymograph (SDS-PAGE with the enzyme's substrate in gel. The proteins are renatured then incubated to degrade the substrate. Staining identifies its location).
The problem is, the substrate dramatically interferes with the polymerization of polyacrylamide gels. I've been trying to use an agarose gel, but its proving to be very difficult (agarose doesn't stick to glass, I may try to purchase frosted glass or plastic plates). Has anyone ever cast a thin, up to 5% agarose gel in a vertical apparatus? Even if I get the gel cast alright, the wells are filled with agarose junk that I'm having a hard time removing.
We use preassembled plastic frames to cast or acrylamide gels, for instance the
Bolt® Empty Mini Gel Cassettes frm thermofischer.
I think this ones can be easily adjusted for Agarose gels.
Hope it helps.
Thanks for the responses and information.
Dr. Spencer,
I'm trying to incorporate lignin. It completely prevents polymerization. I will be trying high concentrations of APS and/or TEMED in an attempt to overcome the issue, but I'm worried that it is degrading the lignin - it is typically degraded through radical depolymerization,
Thanks for your suggestions on the gel apparatus, I may end up trying something similar. As for horizontal gels, I'm doubtful that I would be able to pour any thin enough. I've found that the clearing zone resolution decreases with thickness (currently using 1mm), unless there is a way to load the proteins into a thin horizontal gel?
Hi,
You may try this procedure:
http://www.biotechniques.com/multimedia/archive/00007/98244pf01_7835a.pdf
I did in past and it worked.
However you probably may buy ready agarose gel cassettes for separation of proteins from Invitrogen if I can recall well.
Looks like Lonza is selling that too:
http://bio.lonza.com/uploads/tx_mwaxmarketingmaterial/Lonza_BenchGuides_SourceBook_Section_XIII_-_Protein_Separation_in_Agarose_Gels.pdf
Hope this helps
chris
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