Western Blotting – Why Are My Gels Not Setting?

This question comes up quite often. It can be frustrating having to work to a tight schedule and when for whatever reason your separating and/or stacking gel decides not to set, plans can be sent into a tailspin. 

Usually when a gel doesn’t set, you just start again, but you will be wondering why it didn’t set in the first place. Some common causes are obvious but some are less so.

Clean Your Plates

Those glass plates really need to be clean. If you have residue left over from the previous western run, it can impact on gel polymerization. The same goes for the combs, they need to be cleaned too.

Just before assembling your plates for pouring, spray and wipe (using lint-free tissues) the plates and combs with 70% ethanol.

APS/TEMED

APS and TEMED are important components of the gel mix as they are required for the polymerization of the acrylamide monomers. APS forms the free radicals needed for the polymerization process while TEMED stabilizes those free radicals to speed up the entire process.

APS goes off very easily. If you have made fresh APS and stored it at 4 degrees, it should be fine for about 1-2 weeks (depending on the frequency of use), provided you have not left it to sit at room temperature for extended periods. APS sitting at room temperature for hours should be thrown out.

Ideally, it is better to make fresh APS for each experiment. Just weigh out what you need for each experiment and reconstitute with water.

If despite using fresh APS you find that your gels are setting very slowly i.e. taking hours instead of minutes, you may want to consider using new TEMED.