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storing SDS-PAGE gels for W.Blot


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#1 Shrubal

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Posted 21 June 2011 - 01:16 AM

Hi.

Does anyone know how or if an SDS-PAGE gel can be stored overnight without the protein diffusing so that it can be blotted the next morning? I just realised I don't have enough membrane for my blot and so i'll have to wait till the morning to borrow some... my boss is going to kill me!!

#2 almost a doctor

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Posted 21 June 2011 - 01:26 AM

I think the proteins will diffuse badly if you leave them overnight in the gel. You could maybe fix them with methanol, and then add sds to your transfer buffer to aid the proteins coming off the gel again, but I'm not sure if this will work is just a wild idea. Are you using wet or dry transfer? If dry, you could even use an asymmetric buffer. With sds and without methanol on the gel side, without sds and with Methanol on the membrane side. As I said, just a wild idea.

Can you really not borrow some membrane now? Or just re-run your samples tomorrow? I guess you've used them all... I seriously don't think leaving the gel overnight will work, but I guess if is your only choice you don't lose anything trying.

#3 Shrubal

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Posted 21 June 2011 - 01:34 AM

Thanks almost a doctor

I think the proteins will diffuse badly if you leave them overnight in the gel. You could maybe fix them with methanol, and then add sds to your transfer buffer to aid the proteins coming off the gel again, but I'm not sure if this will work is just a wild idea. Are you using wet or dry transfer? If dry, you could even use an asymmetric buffer. With sds and without methanol on the gel side, without sds and with Methanol on the membrane side. As I said, just a wild idea.

I'm using a wet transfer so i guess your suggestions wouldn't work

Can you really not borrow some membrane now?
Unfortunately everyone is on leave or off for the day

Or just re-run your samples tomorrow? I guess you've used them all...

That's right :-/

I seriously don't think leaving the gel overnight will work, but I guess if is your only choice you don't lose anything trying.

I read some posts from 1995 on http://www.bio.net/b...May/002578.html that gives multiple ways to store a gel before transfer.. i'm gonna try one of these and hope they work



#4 almost a doctor

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Posted 21 June 2011 - 01:51 AM

Interesting thread... I never thought you could store a gel before transfer, but definitely worth a try. As one of the posts says "Probably the only way to know if storing the gel will work, is to just try it, and either it'll work or it won't." ;)

Good luck with it, and post your results here please. I'm actually quite curious about what might happen.

#5 Kaioshin

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Posted 24 June 2011 - 10:46 AM

I've done it before, might have been a small bit of diffusion, but nothing too noticeable/terrible.

I just opened the pieces of glass, left it sitting on one, added a few drops of water, then wrapped it in cellophane and left it at 4C overnight.

#6 Shrubal

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Posted 24 June 2011 - 09:58 PM

Interesting thread... I never thought you could store a gel before transfer, but definitely worth a try. As one of the posts says "Probably the only way to know if storing the gel will work, is to just try it, and either it'll work or it won't." ;)

Good luck with it, and post your results here please. I'm actually quite curious about what might happen.


hi!

so well I took the gel out of the SDS-PAGE buffer and stored it, still in between the glass plates, overnight in the cold room and then proceeded with the transfer the next day and well, I got absolutely perfect results :)But i wouldn't know if I'd have gotten a higher intensity of staining had I carried out the blot immediately [i doubt it though because I have carried out a blot for the same samples before, for a different antibody, and the PonceauS staining was comparable]..So well bottom line is, try and do the blot immediately, but if you have no choice but to wait for a day, it can be done :)

#7 Shrubal

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Posted 24 June 2011 - 09:59 PM

I've done it before, might have been a small bit of diffusion, but nothing too noticeable/terrible.

I just opened the pieces of glass, left it sitting on one, added a few drops of water, then wrapped it in cellophane and left it at 4C overnight.


thanks Kaioshin. I did something similar.. except i just left it between the plates overnight in the cold room

#8 Kaioshin

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Posted 27 June 2011 - 11:45 AM

I prefer taking off one of the glass plates just to be safe, especially if you want to store submerged in buffer. There is the slight possibility of capillary action causing flow through the gel when the glass plates are left together. You guys have apparently shown that that is minimal, but if you ever try this with a lower percentage gel, it could actually have an effect, thus why I suggested taking away one of the plates.

Glad it worked out for you though.




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