immunoprecipitation lysis buffer - (Jan/22/2010 )
Does anyone know what homogenization/lysis buffer I can use to solublize the protein and disrupt protein-protein bonds (ie. I do not want to pull down complexes) but still maintain protein structure integrity and activity. I need to pull down a kinase and assay it's activity independent of other proteins.
You could use a ripa buffer with SDS and then wash several times using stringent conditions (high detergent concentration, high salt concentration).
I think you can do the lysis in 1% SDS and the dilute the sample to 0.1% SDS to do the Ip.
1% SDS won't denature the protein?
Yes, it will.
I can't see a way of disrupting protein-protein bonds while keeping protein integrity and activity, though..
Kinase assays usually permit a simple IP to test protein kinas activity. Can't you use a simple IP to verify?
Sorry, I didnt think of the activity for the kinase assay...
madrius1 is right, and normally to perform kinase assays we do normal Ips
If you need to be sure that is indeed your protein of interest the "active" one in the kinase assay, you could check if you can produce your protein in vitro or in bacteria, and wether is as an active protein (for example the kinase I work with is active when you produce it using an in vitro translation kit), and then use it to do the kinase assay.
(This if you dont want to do a normal Ip)
I'm trying to measure differences in activity between genetically different mouse lines, so I need it pulled out of tissue lysate.
Thanks for all your advice though.
I was just worried about pulling down other proteins that may complex with my kinase that could produce nonspecific activity in the kinase assay. The assay is an ADP glo assay which measures the level of ADP remaining after incubation of kinase+ATP+substrate. I wanted to minimize the possibility that nonspecific substrates and kinases would included in my samples.
I really appreciate all your thoughts! Thank you!