Protein A or protein G for co-IP? - (Oct/31/2006 )
Does it make a lot of difference. My thinking was that protein A=polyclonal and protein G=monoclonal but I notice in the product infor protein G also binds rabbit abs. My protein A beads are on back order until the end of the month. I have either protein G sepharose fast flow or protein A sepharose lypholised. I've read the protocol for swelling the protein A beads but sounds a bit complicated so I'm wondering if I can get away with using protein G beads instead.
It depends on the IgGs: proteinG binds some IgGs and proteinA others. Which proteinA do you have? I have a very easy protocol to resuspend proteinA beads:
-Dilute 250 mg proteinA in 4ml CoIP buffer
Incubate RT in agitation (rotation) for 1h
Spin down 5000 rpm for1 min
Wash with CoIP buffer 3x
Resuspend in twice volume of CoIP buffer
Store at 4ºC
They are protein A sepharose CL-4B beads from Amersham (GE Healthcare) and the protocol on their site has a lot of washing and filtering through ground glass filters etc. Their info on protein G fast flow beads seems to indicate that protein A and protein G have an equivalent binding capacity for rabbit abs (my primary ab) so perhaps they would work? Since I have a big pot of them in the fridge it is worth a try.... Thanks for your protocol, I could try that too!
That is excatly the proteinA we buy and we use that protocol to prepare it.
ProteinG is fine as well but keep in mind it will give you a band around 25 KDa(while proteinA around 50). If your proteins don't have that size you can use it