Leishmania donovani lysate preparation - (Nov/04/2007 )

Dear All,
I am trying to prepare leishmania lysate from one week and i am finding two bands instead of one(major band of tubulin). i have used reagents from my labmates then also i get the same result but my labmates dont. the second band that comes is above tubulin band as detected by ant-tubulin after western blotting. The problem lies in the handling of the lysate. Is is that when adding protein loading dye if there is gap of more than 30 sec before it is boiled then the lysate tubulin binds some protein and it comes as a band above tubulin band that is what i observe in sds-page?
Thanks
Ajay

unlikely. if you are seeing proteins bind to each other while sitting in the sample loading solution (also unlikely) then boiling will break it up (you're not getting covalent bonding).

what are you actually seeing (mw, staining strength of band, etc)?