Hi,

It depends on the type of proteins. This question is very hard to answer. Many reasons can be listed as follows:

1) Maybe your protein is long-lived protein or house-keeping.

2) Primers for RNAi test should be also validated. I don not know if your designed primers cover the cleavage site in mRNA by siRNA.

3) This protein has many homologs in cells. If one is silenced, the homologs will be also supplemented.

Of course, if you provide more details, many people would help your more.

Hi,

I agree with ma. From my perspective the two major reasons could be Isoforms and only one of them is targeted by siRNA and the validation PCR. In addition I would test half life of the protein so you can check how long you need to wait to see reduction on protein level.

Good luck

Sven

I totally agree with Ma and Sven, however if I may add something small and of a quick checking try increase the transduction concentration that you use of your lentivirus particles and do the WB within 24h-48h post transduction. It should increase the silencing and reduce the endogenous protein expression.

Hope it helps.

sometimes there is a substantial disconnect when you measure knockdown at RNA and protein level. qRT-PCR can give eg 99% knockdown, while western blot eg 70% KD. depends on th eprotein, time of assay, many other things. But run the positive control first- to ensure your western blot protocol works fine