how to get specific bands in co-ip - (Jun/18/2013 )
Hi everybody, I'm working on co-ip to get the interacting proteins with my target. but now the problem is no differen between the target antibody and control. I use the IgG and protein A beads as negative control. Everytime, the igG and protein A beads also can pull down many protein bands, nealy have no difference compared with target antibody. i have increse the washing times, but still no help. who knowns how can i continue with it? thanks a lot in advance! Best, Amafantuan.
If you can provide your protocol, that will give us something to troubleshoot -at the moment, there are so many variables that could potentially be going wrong, that there is just no way to tell.
bob1 on Wed Jun 19 04:26:41 2013 said:
If you can provide your protocol, that will give us something to troubleshoot -at the moment, there are so many variables that could potentially be going wrong, that there is just no way to tell.
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OK, that looks fine - how are you detecting your co-precipitated protein? If you are just using coomassie stained gels, you probably won't be able to detect anything. It is normal to have quite a bit of non-specific stuff come across on the beads. For a co-IP it is typical to get about 10% or less of the input protein amount, given the efficinecy of the IP, whether you are precipitating all the directly targeted protein and the stoichiometry of the reaction. In other words - try detecting the protein using a specific antibody.
bob1 on Wed Jun 19 09:05:05 2013 said:
OK, that looks fine - how are you detecting your co-precipitated protein? If you are just using coomassie stained gels, you probably won't be able to detect anything. It is normal to have quite a bit of non-specific stuff come across on the beads. For a co-IP it is typical to get about 10% or less of the input protein amount, given the efficinecy of the IP, whether you are precipitating all the directly targeted protein and the stoichiometry of the reaction. In other words - try detecting the protein using a specific antibody.
I want to use co-ip to find the interaction proteins. Actually it had no difference between specific antibody and IgG, so now I try to do solution digest with total co-ip proteins, then analys with Lc-MS/MS. but no idea wheather it is a practicable solution? thanks! --Amafatnuan
My point is that you probably won't see a difference by eye as the changes are quite small and subtle in most cases. I would recommend MALDI tof-tof for protein detection.