western blot of GFP fusion protein

[EK]

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
>
>
>
>