9 replies to this topic

[mtrnbh]

Hi all,
I had prepared several SDS-PAGE gels without difficulty but recently I have encountered a problem. The gel polymerizes well but when I detach it from clips and pull out the lower spacer, bubbles form between the gel and glass plates and then they come up and become trapped between the stacking and resolving gels; very distorted appearance! I changed every ingredient of the gel but it didn't make any difference. Does anyone know why it happens and what I can do to avoid it?

Edited by mtrnbh, 14 July 2009 - 06:00 AM.

[little mouse]

It already happened to me, but it was not disturbing the migration.

[mtrnbh]

really? even when some bubbles get entrapped between the stacking and resolving gels?  I have prepared 6-7 gels since then and every time it happens I throw it away and make another one :-D! but I wonder why it never happened before?!

[little mouse]

... wait, is it between the gel and the glass, or between the stacking and the resolving gel?
because if the two gels are not separated it should be fine, but if you have bubbles between the 2 gels, then you have a problem

[mtrnbh]

at first they are at the bottom between the gel and the glass but then some of them gradually  come up and stay between the two gels! I really don't know what to do!

[little mouse]

I see.
My bubbles always stayed between the gel and the glass.
I don't know what to tell you,
maybe you should pull the spacer earlier not to "aspirate" the gel. If the gel is more "wet" maybe it's better?

[mtrnbh]

how can I make it more wet?

[mdfenko]

you may be removing the comb too hard. try gently pushing the comb against the cut plate to break the seal between the plates and the comb then remove the comb gently (you may have to push the comb against the cut plate some more during removal).

if the bubbles rose up to the interface of the stacking and running gels then they probably did not disrupt the continuity of the gels. unless your samples are very precious you should try one of the "ruined" gels (if the samples are precious then use standards).

[mtrnbh]

Thank you little mouse and mdfenko for your prompt replies. I'll run one of these gels and see how it will be. Tnx.

[little mouse]

What I meant is that maybe you could try to pull the spacer after only 30 minutes after pourng the stacking gel, when it is not so much polymerized. the gel would be a little more soft and less sticky.
try to pull the spacer very carefully.