Immmunoprecipation question

Martin Offterdinger martin.offterdinger at akh-wien.ac.at
Mon Dec 4 08:22:44 EST 2000


""Jie Sun"" <jiesun65 at hotmail.com> schrieb im Newsbeitrag
news:F157gfUw3ZPpAATnLE700001810 at hotmail.com...
> I thought exactly as you said. but I did the control experiment at first
> using one of my standard protein and ab. The protein concentration is 40
> ug/ml, the ab is polycolonal, I dilute 1:10000 for western and works very
> good. I added 1, 3,5, 8, 10, 15 ul antibody(no dilution) to 800 ul my
> standard protein solution and did the immunoprecipation (overnight
shaking).
> I used 100 ul beads (from pharmacia) to pull down the complex of antibody
> and protein. I ran SDS-PAGE using the sample from sup and beads. The gel
> showed me almost the same amount protein was pull down although I used
> different concentration antibody. Any suggestions?
>
> Thanks!
>
> Jie

This could have two possible explanations.

1) Your antibody has very high affinity and you have to diulte it further
2) Your antigen binds to the sepharose beads (which is rather frequent!!!,
you should always include a control were you add either no antibody at all
or an irrelevant Antibody plus beads.)
regards,
martin







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