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How do you identify protein detected on western blot from a lysate????


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#1 neuron

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Posted 08 March 2011 - 07:46 AM

Hi,

If you have detected some protein on western blot from a lysate, how do you go for identification of that protein?? For example like in SDS PAGE we see the band, cut it then do mass spectrometry to get the identity. But here let say if the antibody is against some probe which binds to a protein in the lysate and you see that protein in the blot but actually when you do silver stain of that gel you don't see the band or you see overlapping bands....so how do you confirm that you are picking the right protein for identification :o ....Has anybody done that??....any suggestions would be helpful :)

Thanks

#2 Adrian K

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Posted 08 March 2011 - 08:39 AM

Hi,

If you have detected some protein on western blot from a lysate, how do you go for identification of that protein?? For example like in SDS PAGE we see the band, cut it then do mass spectrometry to get the identity. But here let say if the antibody is against some probe which binds to a protein in the lysate and you see that protein in the blot but actually when you do silver stain of that gel you don't see the band or you see overlapping bands....so how do you confirm that you are picking the right protein for identification :o ....Has anybody done that??....any suggestions would be helpful :)

Thanks


Hmn... will IEX, plus 2D-Gel electrophoresis helps for your case?
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#3 neuron

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Posted 08 March 2011 - 10:43 PM

yeah ....well thats a great idea..but for 2D..I will have to resuspend my protein lysate in rehydration buffer ..and that will contain urea..I am wondering if that will break the linkage between my protein and the probe? :unsure: ....can you run a 2D in the buffer which does not have any urea, thiourea and CHAPS?????




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