curved gel surface - (Mar/01/2006 )
I have been having a recurring problem with the top of my resolving gel coming out curved with the corners rounded off. It is 11.5%, polymerizes in the appropriate amount of time and I have tried water, isopropanol, methanol and butanol as an overlay. I need the entire width of the gel to accomodate my IPG strip, so I can't just avoid the edges. Any ideas?
Have you mix the resolving gel buffer before pouring?
I swirl the flask with the gel mix 4-5 times after the addition of the APS and TEMED, but don't want to introduce too much air. Have you seen this before with poorly mixed gel mixture?
May be something wrong with the acrylamide.
I think you may need to make fresh acrylamide or buy a commerical acrylamide.
I hope this may help.
this is caused by incomplete polymerization of the acrylamide. give the gel more time. we like to pour our gels ahead of time. you can also "age" your gel overnight.
dumb question, maybe, but are you using fresh APS?
have you checked the pH of your buffers lately?
for better surface, i fill with water all the space between plates. Dilute of course slightely the gel, but the surface is better.
Fred, you fill with water all the space between plates?!
I admit I had a glass of lemoncella, ... but I cannot understand what you are doing?
The space between plates should be occupied by the gel? Do you mean you wet the glass, ... or you ..
you add water after the gel has polymerize to keep it overnight that's it?
Thanks for all of the suggestions. I tried leaving out the SDS and this helped quite a lot. I believe that the 2D protocol at SWISS-PROT excludes SDS from the resolving gel. Does anybody have an opinion regarding how important it is to have SDS in the gel for 2D?
well sorry to all of you, and special regard for missele. I desperately need to be more clear if i want to my post to be at least interesting, if not helpful...
I meant in my post that before pouring the gel, i set up the apparatus and wet the plates, let sit for the time i prepare the mix. The wetting reduce cpapillarity tracts when the gel is polymerizing.
For conservation overnight, i add water+1%SDS in order to reduce SDS dilution.