Abnormal polymerization of acrylamide when preparing SDS-PAGE - (Jan/22/2007 )
Could anyone help me?
The polymerization of the stacking gel seems to be uneven especially in the upper side. It is fluid and cannot forming crystal gel even being rested at RT for several hours. Loading well is misshapen and distorted when removing the comb. But running gel is fine. I used same acrylamide stock to prepare Native Page, it's also fine.
I have checked all buffers, acrylamide/bis-acrylamide stock solution, APS and TEMED, they are OK. Also I prepared new buffers and solutions, there's still same problem.
Anyone has ever faced this kind of problem? I appreciate for any suggestions!
The polymerization of the stacking gel seems to be uneven especially in the upper side. It is fluid and cannot forming crystal gel even being rested at RT for several hours. Loading well is misshapen and distorted when removing the comb. But running gel is fine. I used same acrylamide stock to prepare Native Page, it's also fine.
I have checked all buffers, acrylamide/bis-acrylamide stock solution, APS and TEMED, they are OK. Also I prepared new buffers and solutions, there's still same problem.
Anyone has ever faced this kind of problem? I appreciate for any suggestions!
Maybe you forgot to remove the water (or isopropanol) from covering your resolving gel during polimerization time?
We´re using for 1ml: 0.68ml H2O, 0.17ml 30% Acrylamide mix; 0,13 ml 1.0 M Tris (pH 6.8); 0.01 ml 10%SDS, 0.01 ml 10% APS, 0.001 ml TEMED.
Oxygen inhibits acrilamide polymerization, so I pour ethanol above the acrylamide of the upper gel (although I replace it after some minutes because it evaporates). I think if you leave acrylamide in contact with the air, it won´t polymerize very well.
I agree with Pumuki. But I used 50% water 50% butanol. Does it really matter what to add to avoid air contact? Just curious =)
I have only tried using ethanol, although other people in my lab use butanol. I´m not sure but I think it doesn´t really matter what you use, the purpose is to keep acrylamide protected from the air.
The polymerization of the stacking gel seems to be uneven especially in the upper side. It is fluid and cannot forming crystal gel even being rested at RT for several hours. Loading well is misshapen and distorted when removing the comb. But running gel is fine. I used same acrylamide stock to prepare Native Page, it's also fine.
I have checked all buffers, acrylamide/bis-acrylamide stock solution, APS and TEMED, they are OK. Also I prepared new buffers and solutions, there's still same problem.
Anyone has ever faced this kind of problem? I appreciate for any suggestions!
is this problem new (not happened before with same gel system)?
if not then are you preparing the gelling solution correctly (proper amount of acrylamide)?
what is the acrylamide % for your stack?
are you over agitating the solution prior to pouring (this could introduce oxygen to the solution)?
are you sufficiently washing out your overlay solution prior to pouring your stack?
are you removing excess water after washing out overlay?
Thanks all for replying. Thanks pumuki and mdfenko for your idea and discussion.
I just tried again to prepare the PAGE with using Pumuki's method. It works well. The butanol/water also works.
Another question? Why I have to use the water saturated butanol, not butanol directly to overlay the stack? What's difference?
Thanks again!
Thanks again!
we use butanol directly. some are concerned that it may absorb some water from the top of the gel but we have found no problem.
Thanks again!
we use butanol directly. some are concerned that it may absorb some water from the top of the gel but we have found no problem.
Thank you very much! Your answer is really quick and helpful.
haha... I didnt actually figure out that. Thanks for sharing. Well adding water will save your butanol as well though it is not that expensive.
