Hello everyone,
I knocked out a gene by CRISPR/Cas9 system, and then I confirmed it by WB, there was no protein expression, so I assumed the gene was successfully knocked out, however, when I used the same antibody to do the IF, the protein was in the nucleus, can anyone know how to explain this?
Thanks very much!
I have seen something like this before. Check exactly how and where in your protein the CRISPR/Cas9 made the edit. It could be that it changed the nucleic acid sequence in such a way that made it so that your western blot antibody no longer recognized the protein because the immunogen region was changed so it appeared to be "knocked out" while it really wasn't.
Often times suppliers provide the immunogen region for the antibody so see if it matches the region where CRISPR made the cut.
Also check appropriate controls for IF. Try primary only with no secondary and secondary only with no primary and even use normal rabbit IgG with secondary to make sure the IF results are definitely not any kind of artifact.
Have you looked at IF in the WT cell line? If so does the distribution of the WT protein look similar to the CRISPR/Cas9 edited.
We've been using CellProfiler to output intensities of IF stains and can plot that to detect fairly small changes.
Also have you sequenced your knock out line to see if you cut both copies?
Hi,
Did you use the same antibody for WB and IF??
If not then I would recommend getting the same immunogen for WB / IF
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