loading control in IP? - IP (Dec/29/2006 )

Hi, I am doing IP to look at membrane bound receptors in the rat brain.

when I do western blot, I usually use b-tubulin as a loadign control to see if I load equal amount of protein.

However, I don't know what to do to nomalize my IP data.

How do you know if you load equal amount of protein when you run gels in IP?

I advisor counldn't give me an answer so I need your help.

Thank you in advance, and Happy New Year!

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I suppose B-tubulin serves to normalize your IP data too, as a typical western blot.

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You should blot back for the protein you are IP'ing.

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it´s a question of what you like to show; with IP you enrich a protein (or more precisely: a protein complex) of interest; so precipitated amount depends on various parameters (abundance of protein, dynamics of posttranslational modifications and dynamics of complex composition etc); so it is in the nature of IP that you may precipitate different amounts of proteins; so you cannot normalize your IP´s with an intrinsic protein in Wb; if you separate the whole IP complex in Wb, you may normalize with your extrinsic antibodies to show that you used the same amounts of Ab´s to precipitate; you may normalize your protein sample before IP as loading control in Wb IP; here alpha-actin or beta-tubulin is useful;

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