Gredient PAGE Method

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ss_kalve
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Gredient PAGE Method

Post by ss_kalve » Fri Jan 25, 2008 10:24 am

We do gradient PAGE in our lab. I've problem that firstly we pour resolving gel and ethanol and keep it for polymerization
After polymerization,we get that resolving gel dont in straight line.its level is always raised from middle portion and both the sides having bit low levels.i dont know why.does anybody tell what can we do?This is the picture how we make gradient.
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blcr11
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Re: Gredient PAGE Method

Post by blcr11 » Fri Jan 25, 2008 2:18 pm

This is protein SDS-PAGE, I assume. Two possibilities I can think of. First, there could be leaking slightly around the spacers near the bottom. Best solution for that is just to use a new set of spacers. Sometimes you can "plug" the bottom with a small amount of high % acrylamide gel with a larger dose of initiator/catalyst to make it polymerize very quickly--but you have to be quick yourself or you'll just get a mess inside the gel frame.

Another possibility is the ethanol. It may be fine, but I'm used to using water-saturated butanol. It usually forms a nice, even layer that doesn't evaporate while the gradient gel is polymerizing, but pours off well leaving you with an even gel top. Perhaps the ethanol mixes too well with the gel and/or it is evaporating more rapidly from the edges--though I don't know why it should.

Pre-cast gels are available, for a price.

ss_kalve
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Re: Gredient PAGE Method

Post by ss_kalve » Sat Jan 26, 2008 3:13 am

Thnx for ur suggestion. U r correct but for both the possibilities i got through.there is no probs in 1st step and in 2nd we use 50% butanol.can it be possible that v r using bit old butanol,so it may cause problem?

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