I want to ask some questions of GFP fusion protein western blot?
I have constructed a vector of GFP fusion protein and transfected into
HEK293 cells using lipofectin. I check the protein expression under
Fluorescence microscope 48hr after transfection, it is about 60% cells give
green fluorescence in the GFP control cells and 35% cells in my GFP fusion
protein tansfected cells(localized in nucleus, it is nuclear protein). It is
overexpressed. when I did western blot, I can detect the band of GFP in
control (it is not strong). But I can't find the band of my fusion protein.
I lyzed the cell with RIPA buffer, loaded 10ug of whole cell lysis,
transfered to PVDF membrane. first antibody ( anti-GFP ployclonely
antibody1:1000, santa cruz) and secondary antibody (1:10,000 amersham), ECL
(amersham). the molecular weight of protein is about 120kd(with the GFP
I think the proportion of my protein may be too low in cell lysis to detect
it, maybe I should load more cell lysis. Or I should do the nuclear extract
of transfected cells. Or I should change antibody.
All suggestions are appreciated. thanks in advance!