Co-IP with two polyclonal antibodies - (Oct/19/2006 )

I am attempting to do an IP from SH-SY5Y cell line (human neuroblastoma). My first thought was to try and transfect with the GFP-tagged construct of my protein of interest since I have an IP set up to work with a polyclonal GFP antibody. But these cells need to be in their differentiated neuronal like state in order for the protein we hope to co-IP with my protein of interest to be expressed. And apparently they are nearly impossible to transfect when differentiated.

If I use my antibody against the protein of interest, it is polyclonal and I run into two problems:

1) No antibody to check and see I've IP'd my protein (other than using that ab again)

2) Antibody against the interacting protein is also polyclonal so I will get a heavy and light chain band. I'm not 100% sure exactly where these run, but my proteins are 85kd and 55kd (I suspect I may have this one masked by heavy chain).

My other option is to create a stably transfected cell line with the GFP construct. This eliminates problem 1) but still leaves problem two since I'd most likely use my polyclonal to IP with.

So I am considering cross-linking the primary antibody to the beads? I have no experience of this technique so any advice would be appreciated. What kits work etc.

This is my first week in a new job and I'm coming from a virology background so this is all very new and scary!

Thanks very much,


Rachelle

Hi Rachelle,

1) you can use the same Ab to probe for your protein

2) Also monoclonal Abs have heavy and light chains, respectively migrating as 50 and 25 KDa. There's a new secondary from Jackson Immunoresearch (maybe from other companies as well) that is shown to recognize only the light chain, therefore you shouldn't have problems with the HC band overlapping to the band of your protein.

I would first give it a try in the normal way (which worked for me).