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How to prevent albumin masking of urokinase from conditioned media


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3 replies to this topic

#1 Josip Madunic

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Posted 03 April 2013 - 04:44 AM

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 :)

#2 mdfenko

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Posted 03 April 2013 - 06:39 AM

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

here are a few of the available kits:

pierce

qiagen

proteabio

calbiochem (emd millipore)
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genius does what it must
i do what i get paid to do

#3 Josip Madunic

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Posted 04 April 2013 - 01:41 AM

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

Edited by Josip Madunic, 04 April 2013 - 01:42 AM.


#4 mdfenko

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Posted 04 April 2013 - 04:42 AM

you could use blue dextran-agarose. it is routinely used to remove albumin but we find that it also removes some other proteins.
talent does what it can
genius does what it must
i do what i get paid to do




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