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
Thanks for your reply, but do you have any advices other than commercial kits? Budget-impaired
you could use blue dextran-agarose. it is routinely used to remove albumin but we find that it also removes some other proteins.