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purified protein binds cell surface. how to be sure it is not an artifact?


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#1 pkay

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Posted 31 January 2014 - 10:24 AM

hello there,

 

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.

 

 

thanks!

 

 

 

 



#2 dimensionsbio

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Posted 23 February 2014 - 04:18 AM

Hi pkay.  Further characterization of your refolded protein may help you rule out non-specific binding to the cells.  After refolding how do you analyze the activity of the protein?  You mentioned there is a ligand that binds, but do you have titration curves and affinity calcuations of this binding to compare to the native non-truncated membrane protein?  Also, reduced vs. non-reduced samples by SDS-PAGE, running a native gel or analytical scale SEC can provide data about possible multimerization and aggregration of the refolded proein.  If the refolded protein isn't in its normal state (eg monomer or homodimer), then the binding you see may not be real.






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