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How to prevent albumin masking of urokinase from conditioned media


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#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:

[url="http://www.piercenet.com/browse.cfm?fldID=E0D7C5B3-DC1A-4F13-B19E-E080809BC55C"]pierce[/url]

[url="http://www.qiagen.com/Products/Catalog/Sample-Technologies/Protein-Sample-Technologies/Fractionation-and-Depletion-Kits/Qproteome-AlbuminIgG-Depletion-Kit#orderinginformation"]qiagen[/url]

[url="https://proteabio.com/products/SP-200"]proteabio[/url]

[url="http://www.emdmillipore.com/life-science-research/proteoextract-albumin-removal-kit/EMD_BIO-122640/p_xdCb.s1ORVMAAAEjpxp9.zLX"]calbiochem (emd millipore)[/url]
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#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
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