I am desperated with my WB!! - (Jun/21/2011 )

Hi everybody!

I am working too long with some constructs (maxi quality, they are all ok by sequencing), transfected into COS7 cells with FuGene but I see the same bands into MOCK transfected lane and the transfected ones...one time and one time more...

I have tried many, many, changes...buffers,constructs (GFP and MYC ones)...and ANY ANY ANY change...

All Abs are new, except the secondary one...but I guess this must NOT be the problem (if so, it is too stupid!)...

Does anybody would help me, please???

Thanks a lot

without knowing all the details now, but i can quickly think of some things

1) the promoter in your construct is screwed - try to express something unrelated, from a different plasmid (maybe u did this already e.g. gfp?)

2) what is your transfection efficiancy - are u sure you get the construct into the cells?

3) have u analysed RNA expression of your gene of interest - though i find this rather unlikely, but maybe your protein is highy unstable?

4) try to express your construct in other cells, something simple like HeLa or Hek293

cheers

Is the band you see on the western the band for your protein or is it a non-specific band?

Is there any chance that you have accidentally overflowed the wells when loading your gels?