Low weight protein - (Oct/15/2007 )

Hey guys!


I am having trouble either transferring or detecting my protein. It is about 10Kda and is tagged with Flag. I know that my flag antibody works fine. Any suggestions on how to deal with such small protein?

Thanks

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Use a 22µ membrane, Tris-tricine gels instead of tris-glycine. You can also boil the membrane 5 min, right after the transfer.

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You use "wet" or semi-dry transfer?
I second the use of 0.22um membrane.
Tris-tricine gel would be nice but it's not necessary - I can clearly see 5 kDa on 15% Tris-glycine and get good transfer in semi-dry conditions onto 0.22um PVDF.

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oups, I meant 0.22µ

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Thanks!! I have tried using the 0.22um membrane on semi-dry transfer.

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you can try using a second membrane behind the first in case you are blowing the protein through the first membrane.

you can reduce the current and/or time of the transfer.

if you add a little sds to your transfer buffer, don't. this helps transfer high mw proteins. it is not necessary, and may be harmful, for transfer of low mw proteins.

do you also transfer mw standards? if so, are they prestained? how well do they transfer?

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