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Problems with high molecular weight protein transfer in western blot

western blot

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#1 biochemical mayhem

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Posted 12 April 2012 - 12:55 AM

Hello every Biochemists,
i have a problem regarding the transfer of high molecular weight protein during the wet blot. So, it has been creating problem during detection of the protein by antibody. I could barely see bands of my protein. the recipe of my buffer is :
Tris(25mM), Glycine(192mM) , Ethanol(10%) and SDS (0.01%). For one time I didnt use SDS in the buffer and the blot was better than I expected. If the amount of ethanol has confer some strain to the transfer? Can anyone help me figure this out?

#2 mdfenko

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Posted 12 April 2012 - 11:32 AM

methanol and ethanol are present to strip sds from the proteins as they migrate from the gel to the membrane. sds will interfere with binding. you can add up to 0.05% sds to the buffer to facilitate transfer of high molecular weight proteins. if sds is used then you should add up to 20% alcohol to the buffer.

you don't say if the protein has not exited the gel or if it has not bound to the membrane when you have a poor result.
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