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WB of cellular proteins in human serum


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#1 Dabronzo

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Posted 23 January 2013 - 11:11 AM

I'm working with Human Serum, have had problems trying to clean up the samples from Albumin and IgG, even trying a few cleaning up kits. My goal is to try and find a phosphorylated protein that is usually cellular (eif4E) that might have leaked from bursted cells to the blood/serum. I have found some traces of protein but mostly masked by non-specific binding of what I think It's albumin.
Any ideas of how to clean up these proteins?
Or is there an ELISA kit protocol for working with human serum?
Thanks in advance

#2 bob1

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Posted 23 January 2013 - 11:39 AM

Wow, I suspect you will have your work cut out for you just trying to detect the protein at all - there can't be that many bursting cells normally and given the volume of serum in the body, you might not be able to do this easily.

IgG and other immunoglobulins can be removed by binding to protein A or G beads. Albumin I don't know, but you may be able to try something like a cibacron blue.

#3 Dabronzo

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Posted 23 January 2013 - 11:53 AM

Thank you!
Trust me, I had enought trouble with this WB already, I have got results from a few blots and my PI wants me get this done, so I'm going to try the direct ELISA for this protein and check what I get.
I know the concentration of this protein compared to albumin or IgGs is ridiculous. But the cleaning spinning column didn't work at all.
I got a test kit to try this, I'll post here if I get any good results.




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