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How to prevent albumin masking of urokinase from conditioned media - (Apr/03/2013 )

I am trying to identify urokinase in conditioned media from A1235 glioblastoma cells by Western blot.
However, albumin is complicating things because he dominates in my conditioned media extracts and I am not getting any uPA bands on Western blot, and when I Amidoblack stain my PVDF membranes I can clearly see one big albumin band from 45-65 kDA.
I am cultivating cells prior to extracting conditioned media, 6 hrs in serum-free media. Should I increase that time to get rid of albumin or do you know any other way how to prevent albumin interfering with my urokinase identification. Much obliged :)

-Josip Madunic-

you can use an albumin depletion kit (antibody based are more specific than dye affinity based).

here are a few of the available kits:




calbiochem (emd millipore)


Thanks for your reply, but do you have any advices other than commercial kits? Budget-impaired

-Josip Madunic-

you could use blue dextran-agarose. it is routinely used to remove albumin but we find that it also removes some other proteins.