How can i dual with wrong size band? - (Dec/05/2008 )

Dear all,

Recently i am blotting a protein which has a size of 36kda. After a few trials, i can only get bands at around 50kda. I incubated the protein lysate in sample buffer (with b-mercap) at 95C for 5min, is it enough to break all the protein-protein interaction? What else can i try?

Thanks

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SDS-PAGE is a relatively qualitive method to measure protein size. It is normal to get bands at not quite the right size, although what you are seeing is quite a discrepancy. Your boiling step is fine, and should eliminate cysteine-bond mediated interactions. Is this a purified protein with some kind of tag (His, GST, etc.)? That will add weight and should be considered.

There may be other interactions which are not eliminated in reducing buffer, which may be worth looking into as well.

If you have cloned this protein though, I would sequence it immediately. If there is a frameshift, your stop codon may not appear at the proper spot, resulting in a lengthier protein. This has happened to me (although mine became truncated rather than elongated).

Also -- what are you blotting? They don't happen to be Staphylococcus lysates, do they? Staph has a ~50kDa Protein A which binds all IgG molecules, thus producing bands at that size. I believe other bacteria have similar proteins, but Staph is the most characterized.

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