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I am desperated with my WB!!


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#1 Lightblue

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Posted 21 June 2011 - 07:49 AM

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

#2 Bomber

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Posted 21 June 2011 - 11:08 AM

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

#3 bob1

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Posted 21 June 2011 - 05:17 PM

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?




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