Problems with my co-IPs, please help me!!

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la biologiste
la biologiste's picture
Problems with my co-IPs, please help me!!

Hello!

I hope that someone can help me.

I work on a endothelial transmembrane protein (she is a cadherin and she weights 125 kDa ).
I want to know if an another protein can interact with her (a 130 kDa protein).
So I did a co-IP. I use a polyclonal antibody which immunoprecipitate the 130 kDa protein and then,I probe my membrane with a monoclonal antibody with reconize my 125 kDa protein. I find a lot of problems with my co-IP.
First I think that my G-sepharose beads alone can interact with my 125 kDa protein (I use the protein G sepharose 4 fast flow of GE healthcare.)
My lysat with beads showed bands on my membrane.
I tried to saturate the beads with 3% of BSA, but in the case of the lysat only or in my negative control (IP with a IgG non revelant) I have bands.
Even when I precleared my lysats with beads I have the same problem of course!!

Have you ever seen this problems and have you got some solution ?

Thanks a lot for you help!! (sorry about my proximatly english ;) ).

Bye and have a nice day

alice

kriodos
kriodos's picture
Unfortunatly protein G binds

Unfortunatly protein G binds very effectivily serum albumin (like bsa) and others proteins. then it can bind something in the lysate. Other problem can be the conditions on your western blot. If are too permisive your antibodie can recognise others proteins did you do a wester with the hole lysate for see if you get only a band of 125 KDa of molecular weigth? if not, do it. If you get more bands than you want, change the western conditions and them repeat te Co-IP experiment
Bonne Chance