I am desperated with my WB!!
Posted 21 June 2011 - 07:49 AM
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
Posted 21 June 2011 - 11:08 AM
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
Posted 21 June 2011 - 05:17 PM
Is there any chance that you have accidentally overflowed the wells when loading your gels?