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SDS-PAGE with 500 ug protein


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#1 krithbala

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Posted 28 March 2011 - 03:57 AM

HI

I am trying to perform a 2D analysis with 500 ug of protein (12% PAGE). I know that the protein to SDS ratio has to be 1:1.4 Can I increase the SDS in the resolving and stacking buffers since I cant alter the amount at the sample stage? Please help. Krith

#2 mdfenko

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Posted 28 March 2011 - 12:39 PM

you shouldn't need to increase the sds in the buffers.

have you tried running with the normal formulation? if so, what happened that makes you think you need more sds?
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#3 krithbala

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Posted 29 March 2011 - 08:23 PM

I have tried running similar samples on 1D and found that they didnt move much. The bands remained at the top as blobs, right after the stacking gel front. I thought adding more SDS to the gel buffers would help me with the problem in 2D.

#4 mdfenko

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Posted 30 March 2011 - 06:55 AM

what is the expected molecular weight range of the proteins?

are you sure that 12% is not too restrictive?

have you tried a lower acrylamide concentration or a gradient?

are you sure that the protein is solubilized? did you boil in sample buffer?
talent does what it can
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i do what i get paid to do




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