western blot of GFP fusion protein

Rene renevanwezel at hotmail.com
Fri Oct 24 04:20:52 EST 2003


I'm having similar problems with plant material, I assume with control
you have free GFP?! Anyway, fusion protein generally lot less, and
additionally some mutants break down more easily, resulting in more
bands.
Have you tried antibody for the protein itself?

Rene.


"EK" <nobody at elnino.com> wrote in message news:<r7Tlb.27$_4.8639 at news.uchicago.edu>...
> High MW proteins are much slower to transfer. Most probably, in your case
> you are not transferring any at all. Try longer transfer times. Some people
> posted here before that supplementing the transfer buffer (Tris-Gly-20%MeOH)
> with 0.05-0.1% SDS helps, too. Also, check if you are properly inhibiting
> proteases in your lysates. Adding peptide inhibitors, EDTA and PMSF would
> help.
> -Emir
> 
> "tiger" <zhuhu at cuhk.edu.hk> wrote in message
> news:bn8phr$nq2$1 at justice.itsc.cuhk.edu.hk...
> > hi, anyone: 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
> >
> >
> >
> >



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