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Concentration of secreted protein - ...step before IP.... (Apr/27/2006 )


I've already send a message today about co-IP protocol of native protein extracted from "normal cells", but I need some other advice!!

I'd also perform IP of secreted protein, and I'd like know if I can directly incubate my coupled Ig-Magnetic beads with the culture medium and just check for the correct pH.
In other way, I'd prefere concentrate my sample in one hand to increase the amount of precipitation protein and in other hand to avoid immunoprecipitation of serum protein. In this case could you advice me about a protein concentration protocol compatible with IP step....

Thanks for your help...


So, if I am getting this correct, you are pulling down your protein from an IP bead that is cross-linked with antibody to your protein. You would like to get the concentration of your eluted protein without contamination of serum proteins?

I did this opposite from you. I used a PEG precipitation to get rid of serum globular proteins first. After that, I ran my supernatant onto an IP column that was cross-linked with my antibody (so that my antibody didn't elute with my protein!). I washed the bound protein with buffer to remove most other contaminants. Then, I eluted my protein from my column. Your protein solution may contain contaminants at this step also, but are visualized on a gel. Any Bradford or BCA will take into account all of your proteins in solution. Continue with purification techniques until your protein is as pure as you need it.

If you are so lucky that you have a great monoclonal antibody, you can run on a western blot - increasing amounts of known protein and increasing amounts of your protein - transfer onto membrane, blot for your protein and do a comparison quantification by judging intensities. However, I wouldn't try this with a polyclonal.