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IP: specific bands in control lanes

IP western blot IgG

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#1 noliwatson

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Posted 13 November 2013 - 03:06 PM

I was hoping someone might be able to help me troubleshoot some IPs I have been doing. In my IPs, I detect very clear bands of the appropriate size in my pull-down lanes. Unfortunately, I detect identical bands in my IgG control lanes. Protein of interest X is pulled down with an endogenous antibody; protein of interest Y is HA-tagged and pulled down or detected using a high-affinity rat HA antibody. I can do the IP either way (IP X, IB Y; IP Y, IB X) and still detect specific bands in IgG lanes. Protein X does run very close to IgG heavy chain (about 54 kD), but can be resolved on a 16 cm gel. Protein Y runs about 37 kD and has a distinct pattern of three bands which is quite unmistakable (staring at me from my control lanes.) I have ruled out contamination by replacing all my reagents and using several companies' beads (protein A/G or protein G) and control IgG (Millipore and Bethyl, both rabbit.) I have even used mutliple antibodies to protein X for pull-down and Western. One is a mouse mono, one is a goat poly. There is one negative control that does work: a "no antibody" control in which I incubate lysate with no antibody, add beads, and elute it alongside my other samples. This runs clean or with the faintest of background bands. I always run a direct Western/input sample alongside my IP samples to confirm expression of the proteins at the right size. I am at my wit's end and I would appreciate any help!



#2 CarrieX

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Posted 01 January 2014 - 12:24 PM

Hello! I am also doing coIP right now. Did you solve your problem? What do you mean by the control IgG? Does it the preimmune serum or a nonspecific IgG as negative control?







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