i purified the extracellular domain of a transmembrane protein to check for unknown protein ligands. after some trouble with purification (refolding!) I tested my protein for cell surface binding. i use flow cytometry to check for binding. on some tumor cells and primary cells i get some nice signals (high MFI; lots of positive cells compared to controls)....too nice in my eyes. i am especially suspicous as there are cells among the tested which should not (according to literature...) bind my protein.
This is my protocol for staining:
1)wash the cells twice
2)+ 3 µg of my Protein; 30 min 4°C
3)wash; + anti-His or protein-specific AB; 30 min 4°C
4)wash; +secondary AB (PE or APC labled); 30 min 4°C
controls: 1)cells+secondary AB only; 2)cells + first and secondary AB; 3)cells + his-tagged DHFR+first and secondary AB --> all negative for PE or APC fluorescence.
buffer for washing and incubation: PBS + 5 % FCSi
perhaps important: protein is stored in 25 mM Tris Buffer + 1 mM DDT; 1 mM EDTA at pH 8; host for expression e.coli
the protein has an exogenous ligand (not of protein origin) which is bound effectively so i guess the protein is properly folded. additionally i am planning to do some pulldown assay and dot Blots/far-westerns to check for ligands. but i think my flow cytometrc approach is much more elegant as it leaves cells intact.
so any suggestions how to rule out i am seeing artifacts in the cytometer? i am planning to do some pulldown assay and dot Blots/far-westerns to check for ligands.