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-   -   problem with IP for virion protein with human IgG (http://www.molecularstation.com/forum/immunoprecipitation-forum/56978-problem-ip-virion-protein-human-igg.html)

hfe0 02-15-2009 04:46 PM

problem with IP for virion protein with human IgG
 
Hello, I am trying to work out the IP of virion protein with human IgG.

I tried to pull down various conc of purified virions with human IgG with added background H293T cells in either a PBS buffer or a NP40 buffer using protein A plus beads (Pierces). I use glycine PH2.5 buffer to elute. I got all same Ct values for a real time PCR assay afterwards for 0, 250, 25000 copies of virion although it appears the background Ct is equivalent to 250 copies.

Why my non-viron control also got Ct on real time assay? (I have tried many times so it can't be contamination all the time)
Why I did not see pull down for them as 0 to 25000 copies of virion got the same Ct? I also see high background.

the virion and human IgG have been confirmed in the ELISA. I can't make it work. So hope anyone here can give a hint.

Thanks

hfe0 02-18-2009 01:54 AM

Re: problem with IP for virion protein with human IgG
 
I increased my input of virion protein and was able to see the binding. However it looked that most virion protein was not bound and in the flowthrough.

What other buffer can I try? I tried RIPA and PBS+tw.


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