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Extra band in western blot - (Jun/24/2006 )

Im running western blots for HSP 70, HSP 90 and HSP 27 in pig oocytes. I wash in PBS-T and block in PBS-T + milk. The primary concentration is 1:500 (mouse) and secondary 1:10,000 (goat).
Each western produces an extra band at approx 60kDa with each HSP antibody, which are all run on separate gels (i.e. HSP 70 on gel 1, 90 on gel 2 etc).
Any ideas what could be the problem?
Also, what are the best lysis methods available?


I had the same problem with an anti-p63 antibody: I had a band at 50 KDa and never understood what it was. Sometimes it happens. I learnt to live with that.
There are several lysis methods, more or less harsh. The choice depends on the experiment you need to do. Some buffers contain SDS, some contain urea, some none of them. Some methods (when you want to do a CAT assay for example), are based on freezing and thawing the cells previously resuspendend in PBS. Sonication, passage through a siringe needle; nuclei preparation and lysis of nuclei...