Co-immunoprecipitation of phospho-proteins - I"m looking for a sensitive protocol to maintain the phosphorylate (Sep/24/2007 )
Hi everyone,
I'm about to start co-immunoprecipitations of cell lysates for a protein which possibly interacts with phosphorylated ASK1.
I'll do the IP with the non-phosphorylated protein antibody and the WB with the phosphorylated protein antibody for financial practicallity. My concern is to maintain the phosphorylated state of ASK1 through the IP.
Has anyone done this protocol before? Or is smart enough to determine the likelyhood of dephosphorylation of ASK1 proteins during a standard IP protocol.
My literature search found several papers that have done standard IPs on phospho-protiens, but given significant differences in the WB protocols -in particular the use of 5% milk as a blocking agent in their protocols compared with our 5% BSA block to negate the de-phosphorylating effect of casein in ours, which suggests a greater sensitivity in my samples; I'm reluctant to proceed with their basic IP protocol.
Any ideas?
cheers kat c
Australia
I'm about to start co-immunoprecipitations of cell lysates for a protein which possibly interacts with phosphorylated ASK1.
I'll do the IP with the non-phosphorylated protein antibody and the WB with the phosphorylated protein antibody for financial practicallity. My concern is to maintain the phosphorylated state of ASK1 through the IP.
Has anyone done this protocol before? Or is smart enough to determine the likelyhood of dephosphorylation of ASK1 proteins during a standard IP protocol.
My literature search found several papers that have done standard IPs on phospho-protiens, but given significant differences in the WB protocols -in particular the use of 5% milk as a blocking agent in their protocols compared with our 5% BSA block to negate the de-phosphorylating effect of casein in ours, which suggests a greater sensitivity in my samples; I'm reluctant to proceed with their basic IP protocol.
Any ideas?
cheers kat c
Australia
if it is a commercially available Ab, you may have the protocol recommended by the manufacturer; to save phosphorylation, use a phosphatase inhibitor cocktail in parallel with a protease inhibitor cocktail
by the way, do you use an anti-phospho ASK1 Ab? I am looking for a well working one...
at least you should add NaF to all your solutions. Cheaper than the cocktails ad worth.