IP a protein from human serum - (Oct/19/2006 )

hi
I want to IP a protein from human serum, but I have no idea of how many sample and antibody I should load to start. I´ve asked to the brand from which I bought the IP kit and they replied me that I just must try to determine that empirically, but I don´t have any reference since I´ve never done an IP and for human serum I can´t find any ideas (all that I find is for cell lysates).

My ab is polyclonal. I know it will have to optimize the IP trying different amounts of both sample and Ab, but I don´t know how to start. I don´t want to spend a lot of my expensive Ab trying different amounts

And another question: should I remove albumin to enrich my serum in the lower abundance proteins? I think this is a good idea, isn´t it?

thanks

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the limiting factor are mostly the amount of antibody for IP; how do like to perform precipitation? supposing you take protein A/G, it is highly recommended to clear up serum with pre-incubation with protein A/G; as there should be some immunoglobulins in serum, do not save protein A/G, and apply clearing at least twice; if you can estimate the concentration of your protein of interest, you may be able to roughly calculate the stoichometry of Ab: protein of interest; if it is not possible, you may choose amount of serum in excess to Ab; incubate O.N. with gentle rocking, or better over-head-cycling at 4°C in the cooling room; percipitate with protein A/G; for Ab:protein A/G antibody manufacturer should provide a protocol

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My Ab is polyclonal (I have 100 uL of whole rabbit serum), so I chose protein A. The datasheet says that I must prepare a determined amount of purified Ab, but I don´t understand because polyclonals are not purified, or am I wrong?
Another guy from a lab told me that he used 100uL of sample (cell lysate) mixed with 1 uL of Ab. I know this may be different depending on your Ab and sample, but could be a good option to start with?
Thanks for the idea of clearing up serum with pre-incubation with protein G, I feel so inexperienced!!
I attach you the protocol provided in case you can have a quick look. LOTS AND LOTS OF THANKS

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the manufacturer´s protocol is global but for optimum results your specific system has to be optimized;

polyclonals are mostly provided affinity purified (it should be a clear solution) or as serum (you know how serum looks like); specific concentration should be declared, or ask the manufacturer if there is sth not clear;

to start with 1 µl depends of course on concentration; but I think so a low amount is only useful for optimization and would only be enough for 1 gel slot to check IP; if you need much more immunoprecipitate, I would start at least with 10 µl

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OK, Thanks again kosmodrom.

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