Hi everyone,

I have done gene knock down by shRNA and also siRNA. After transfection, in RNA level I have effiecient (80%) knock down but it is not possible to see KD in protein level by western blotting and I do not have any KD in protein level (same experiment). And also, I confirmed the efficiency by checking RNA level of some direct and indirect targets of my gene.

what could be the reason of this problem? Does any body have any suggestion?

I think KD worked, because I see the effects in some direct/indirect targets (in RNA level).

I do not have antibody for those direct/ indirect target genes to check them in protein level as well but for all known targets I have strong effect after KD.

antibody is specific (two different antibodies from different company for that specific protein).

shRNA and siRAN are 100% specific.

primers for all targets are specific and efficient.

* Even I did experiment in linger time to check whether half life of protein is long, but I could not see any changes. only only one time I saw efficient KD in protein level. And after that I could not get same as first time. Most of the time I see KD in RNA level.

* I checked many of possibilities like different trasfection reagent, amount of plasmid using for transfection, time of experiment, antibody from different companies ....

Thanks in advance for ant help!!!