Sequential IP - (Jun/29/2007 )

Hi all,

I'm a rookie for the protein field, could anyone tell me when I should use sequential IP instead of co-IP?
I did get a lot of background bands in my co-IP result, could sequential IP improve this?
Now, we suspect that there are three proteins binding to each other in my cells, can I use sequential IP to prove this?
I would be highly appreciated if anyone could kindly provide me some sequential IP protocols.

Thanks!!

Elaine

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your point is not really clear to me beside that you like to circumvent high background;

co-IP has at least 2 proteins in focus; ideally you precipitate each one of it with an appropriate Ab and should found in each precipitation both proteins (2 IP´s);

if you try a to characterize a ternary complex and work with 3 Ab´s for each protein, do 3 IP´s which are of course performed sequential;

you need a different set of the 3 Ab´s for the immunoblots

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