Problem with acrylamide gels
Marek Budzynski
Tuesday, 11 August 2009 15:34 UTC
Hi.
Recently I have a problem with acrylamide gles.
Whenever i try to load samples on the gel, the samples run out because clogs in the wells. These clogs are in even fresh gels and they cannot be removed by clearing wells by syringe.
Does anybody have got these problems or knew how to remove avoid these clogs?
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Replies
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Hi,
Check whether you are using same size of of plate & comb for making gels.or try to increase the amount of your loading buffer.. are you using high % acrylamide gels?
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Dear Marek,
Do you know the nature of the clogs? Would these be polymerised acrylamide?
Did you check if you changed any of or solutions recently?
Are you using to much APS?I cannot imagine how to remove the clogs, but to avoid them I would use good qualitity acrylamide, bi-dest water, and adequate amounts of APS and TEMED.
In addition, I would ensure that the gel is not polymerising too fast, if necessary, cool a bit your solutions prior to poor them. But pay attention that it will take longer to polymerise.If none of these work, I would simply prepare new solutions (buffer, acrylamide, water and APS).
I hope you’ll solve your problem.
Cheers,
Ana
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I used to clean the wells in sequencing gels by sucking out the excess acrylamide with vacuum pump. If you have urea in the gel try to clean the well immediately before loading the sample, because in 1 minute urea will fill up the well again.
But I don’t like that your samples “run out” – that means the density of the samples is less than that of the buffer in the wells. This can happen if you use wrong loading buffer (for example, buffer for native gels in denaturing gel) or by mistake take 10x running buffer instead of 1×. It’s stupid, but happens sometimes:) -
Anonymous
Hallo,
I have a small question about the imidazole/zinc sulfate dye of 2D-SDS-PAGE gels.
I am trying to do that and according to a pubblication (Castellanos, 1995)I have read that it is possible to do it in double staining (Coomassie-imidaz/ZincSulfate)…I have used Coomassie stained gels and unfortunately with the reverse staining I could detect only the bands corresponding to Coomassie (the look trasparent of course) but actually nothing more….I have followed the procedure as they have described….maybe have I to de-stain the Coomassie gels, before the negative staining to see even the negative proteins?Thanks in advance for your help :)
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