possible problem in western blotting/sample preparation?? - (Jun/15/2005 )

Hi there,
I am trying to isolate a recombinant protein from mammalian cells. I tagged this protein with HA11 and FLAG tags to immunoprecipitate it from mammalian cell extracts but the protein does'nt express very well, (this protein is a GPCR), which i checked by a flow cytometry based assay. To overcome the expression problem, i tagged the protein with GFP so that i could visualize the expression live. Now this protein expresses very well (i can easily see this protein in a fluorescent microscope with > 80% transfection efficiency in COS-7 cells), but i still cannot isolate the protein. On a western blot, the recombinant protein (anti-GFP reactive and SAM11 (i.e. endogenous protein recognizing antibody) reactive bands never show up. Only the endogenous protein bands show up. Because of this i cannot even IP this protein even though i know it's getting expressed well enough.
I've used different lysis buffers for lysing my cells, and tried different batches of antibodies but no use.
I think that maybe after lysis of the cells, the GFP tag is screwing up the conformation of the protein such that it is rendered unrecognizable on a WB. If so, can anybody advise me how to get this protein to show up on a blot & eventually isolate it??

Stop lysing and start sonicating the cells.


Also, dont boil your samples and see what happens.

Hi,
i've tried the non-boiling of samples idea based on the presumption that boiling might aggregate highly hydrophobic membrane proteins, instead heated the samples at 60 C for 10 mins, but still the reqd. prot. bands did'nt show up.
Will try to sonicate the samples instead of lysing them this time & then running the lysate & seeing !!!!
Thanx anyway,

Hi,
I tried the sonication of samples instead of lysing them with detergent only and it still does'nt show up on the western blot. Am very confused about what's going on??
any help would be appreciated......