Hi,all
I did shRNA KD in Jurkat cells, the KD efficiency is 80% in mRNA,but western results always showed unchanged protein in the KD group, and also, I have KO mice of this gene
(by CRISPR technology),the genomic PCR showed KO band very clearly ,but protein level detection always showed similar protein level compared to WT mice, this is really confusing,I wonder if anyone can offer me any suggestions.
I could think of 2 ideas:
shRNA: Does your protein of interest have a very long half-life? It could be that you perform an efficient knockdown of the RNA however the protein is very stable and therefore still visible in your western blot. You could try to increase the timecourse after the knockdown.
CRISPR-KO: Where does your gRNA cut? It can be that, if the cut is in the 1st or 2ndexon of a gene that an alternative start site (ATG) is used by the polymerase wherefore a shortened protein will be generated. Try different sgRNAs, check for an INDEL with a clear frame shift and check them on your western blot.
Hi Xia Liu
"I did shRNA KD in Jurkat cells, the KD efficiency is 80% in mRNA,but western results always showed unchanged protein in the KD group, and also, I have KO mice of this gene"
It is not uncommon to notice such a case, because knock down can not show its effect on protein levels at the time it did on the mRNA expression.
So, you have first to draw up a time line such as (12, 24, 36...h) and detect the point at which you have the lowest expression and take a look at the expression trend. Then you can can choose a point where you would get low mRNA and protein levels either.
The knock down efficiency varies according to the cell/tissue type. This may range from 36 to 72 h (in my experience) was enough to achieve a lower protein levels, but again this will depend on the type of cells you with.
As the previous answers, how many days of KD did you do? It depends on the protein turnover lifetime of the target of interest, but I would recommend continuing to KD for 2-3 days after the mRNA drops.
And simply, is the specificity of the antibodies you used guaranteed by KD or KO? There are some terrible manufacturers who sell their antibodies just because they see bands of similar molecular weight.
Christoph Neumayr Thanks for your suggestion. For the KO mice,this gene only have one exon and 192aa,so I don't have too much choice for the exon selection.I am thinking is it possible that the protein is hard to degrade in KD cell line but already dysfunctional, but still curious why the KO mice also show stable protein level even the genomic results show ko of the exon area?
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