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wester blot of GFP fusion protein

Eric say.no at spam.now
Thu Oct 23 21:01:22 EST 2003

tiger wrote:
> hi, everyone
> 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 tag).
> 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!
> mike

Do you have a protease inhibitor cocktail in your lysis buffer?  This could
explain the low yields on your western blot.  You may also want to try
carrying out your lysis steps on ice which can also improve yields.  How
much total protein did you load on the gel?  You can always run tittered
lanes on your gel to find the optimum loading conditions for your cells and
antibody, e.g., run 10, 20, 30µg or whatever gradient you think is
appropriate.  Are you getting any non-specific bands showing up on the blot?


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