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Stacking Gel Polymerization - (Mar/31/2011 )

My lower resolving gel for western blots has always turned out well. My upper, stacking gel, however, continues to fail me. When I insert the comb, there are no bubbles. When I come back to the gel 30 minutes later and it is polymerized, the gel has completely pulled away from the comb, ruining some of the wells. I understand that this is natural to an extent but sometimes only four of my wells are viable. Any suggestions to solve/manage the problem? How do I get the gel to 'shrink' less or at least in an upward fashion so the bottom of my wells are usable?

-MarieM-

Typically when I am preparing my gel I use a slightly higher ratio of TEMED:Polyacrylamide for the stacking gel than I do for the resolving gel. For example if your recipe for your gel calls for 2.5 ÁL, maybe increase it to 3 ÁL for the stacker. This will help your stacker polymerize a little faster and perhaps prevent those unpolymerized regions from forming. Make sure you put your comb in as soon as you poor it, since oxygen will inhibit polymerization. You could also let the gel polymerize a few minutes longer, sometimes those regions will eventually polymerize.

-proteaMatt-

I've had some issues with my stacking also, lately. Although it often polymerizes quickly, it sometimes "fails" to polymerize quickly enough and the last well is ruined. I found that bringning your reagents to RT and using really fresh APS helps to prevent this, although not being fail-proof.

You could also try to use freshly made acrylamide, and verify the pH of your buffers.

-madrius1-

We always store our reagents at room temperature.

-proteaMatt-

MarieM on Fri Apr 1 05:22:02 2011 said:


My lower resolving gel for western blots has always turned out well. My upper, stacking gel, however, continues to fail me. When I insert the comb, there are no bubbles. When I come back to the gel 30 minutes later and it is polymerized, the gel has completely pulled away from the comb, ruining some of the wells. I understand that this is natural to an extent but sometimes only four of my wells are viable. Any suggestions to solve/manage the problem? How do I get the gel to 'shrink' less or at least in an upward fashion so the bottom of my wells are usable?


Hi,
Try this protocol!!!!

for 5ml:

2.9 ml H2O
1.25 ml Upper Tris
0.85ml Acrylamide
25ul APS (Please vortex APS when u take from -20 (u should mix well anything u take from -20/-70))
10ul TEMED
hope it will works..
gud luk...

-MAGESH-