western blotting - (Jun/02/2007 )
Hi, there. Actually I am detecting Rb protein in Western blotting, from now I can see the band using BD (G3-245) ab but I can not detect it recently. I do not understand why it is not working now. Do you have any advices to me? I am using semi-dry (bio-dad) and criterion gel and invitrogen gel sometimes. Moreover I am using the 20% MeOH in tris-glycine buffer for transfer and 25V/90mA condition for 90min. thanks
blotting efficiency can be checked by Ponceau S staining of the Western blot; in the case of inconsistancy, try tank blotting for more homogeneous protein transfer
Rb is a nuclear protein, nuclei may be insufficiently lysed?
do you have also a anti-phospho Rb antibody to use?