Western Blotting for Large Proteins - (May/08/2007 )

Hi,

I want to run a western and probe for a protien that is 600 kDa but I am not sure how to do it. I usually use precast gels from invitrogen with MOPS running buffer but my molecular weight marker only goes up to 250 kDa. What kind of gel should I use and what kind of running buffer and marker? Also, is there anything specific I should do for the transfer?

Thanks.

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You should make your own SDS-PAGE gel and try with different concentrations of acrylamide, that will give you some flexibility. Any protocol will be good. I do it in standard Tris-glicyne buffers. For 600 kDa, I'd try first 5% gel, at least 1.2 mm thick. You should buy a marker which has the 600 kDa band. Maybe run the gel long enough to make the smallest proteins escape.
The transfer, I'd make it longer, 70 minutes in 100 V instead of my regular 60 in 100 V. Afterwards, dye the gel with Coomassie blue to check efficiency and/or the membrane with Ponceau S (it washes easily, and you can still probe the membrane with antibodies)
Manipulate with times and concentrations, after several tries you should have it set.

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we use to separate large muscle proteins in self-cast gradient SDS gels ranging from 2% to 14% AA; efficient blotting may be performed in a tank O.N.;

marker is a problem; we use purified titin (~1000 kDa) and myosin HC (~200 kDa); if you definitely know the molecular mass of your protein, and as even in crude extracts rarely other proteins of 600 kDa are abundant, your protein of interest serves as a reference of its own;

good luck

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