2 replies to this topic

[lrispoli]

Wanted to get some opinions from the experts in immunoprecipitation. Have cell lysates from samples that correspond to samples used for microarray analysis and would like to "validate" results have seen. I.e. if mRNA was found to be increased four fold with treatment was the protein also. Specifically wanted an opinion how feasible is it to run multiple immunoprecipitations on a single lysate (or even has it ever been done, could not find anything with recent searches). Of course we are on a budget so looking for economical methods to get the most bang for the buck. Plus samples are small so doing straight westerns and stripping multiple times likely will not be the best idea. I was hoping to be able to immunoprecipitate multiple proteins in single step but of course that would depend on that the proteins are of distinct different sizes for the western. So the only other alternative is multiple runs for each sample. So let me know if I am crazy or if this is even feasible.

Thanks

[molgen]

lrispoli, on Feb 23 2009, 02:10 AM, said:

So let me know if I am crazy or if this is even feasible.

It has been done before. But, as you wrote, the proteins must be of deferent sizes.
Or, much more tricky, if you use different AB you might be able to bind the different AB to a different precipitant (look at ABcam catalog for a table of witch AB binds to witch proteins a/g).

Edited by molgen, 23 February 2009 - 04:36 AM.

[Dr Teeth]

If the proteins are of different molecular weights, why not do Westerns where you cut the membrane horizontally using markers?  This way, you can stain for each protein at the same time with one sample without stripping each time.  Or, use antibodies with different secondaries simultaneously.