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Western Blotting of Proteins less than 10 kDa

western blot small proteins

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#1 scichem

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Posted 16 November 2011 - 06:54 AM

I'm trying to determine if my mammalian system is secreting my protein into the media. I am doing this through the use of a western blot. I am first separating using 18.3% Tris-Tricine gels to get good separation of the small proteins, and a pre-stained MW marker to visualize where my protein should be on the gel. My protein is ~ 8kDa so I am worried about it blowing through the membrane. I have been using Immobilon membranes with a pore size of 0.2 um. What do you recommend for transfer time for a wet transfer? I am using a Biorad Criterion Blotter system. When I hook it up to run, it doesn't allow me to go higher than ~35V which shows around 450-500 mAmps. Any suggestions for transfer time and voltage/amps conditions? If my MW marker ~10 kDa transfers to the membrane, my protein should have as well, correct?

Thanks for your help!

#2 mdfenko

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Posted 21 November 2011 - 09:26 AM

in answer to your question, yes, if you transfer the 10 kDa protein then the 8 kDa should have transferred.

what is the size range of the proteins from which you are separating the 8 kDa protein?

a 10% tris-tricine gel should be work well with 8 and 10 kDa. and transfer should be easier.
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