Western Blotting of Proteins less than 10 kDa - (Nov/16/2011 )
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!
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.