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Western blot question - (Jun/14/2004 )

Hi all, laugh.gif

I am expressing a recombinant protein in yeast. The protein of interest is composed by a heavy and a light chain. When I detect it by reducing conditions western blot I can see both heavy and light chain bands (I have used two different polyclonal Abs for detection with same results).
The problem is that there are more than one band below that corresponding to light chain.
I think it may be triggered by degradation. However I would like to know other possible causes.

Thanks a lot for your appreciated suggestions!



If the sizes of other bands are always the same, then highly specific processing might be occurring. The objective of this processing (C or N terminus) could be to send your protein to the membrane or any other destination for no apparent reason. Check cell free supernatant and clarified cell lysate separate from the total lysate and see if the Western pattern changes.