I've been making CRISPR knock-out cell lines of my protein of interest. Unfortunately, there is no commercially available antibody to check the presence of my protein. The only method I have been using so far to check for knock-down is qPCR.
However, I have encountered a problem after making my CRISPR knock out cell line. qPCR did not detect the change in the mRNA level. This is what I was worried about since CRISPR makes faulty gene leads to no protein production, however, the faulty gene can still be transcribed into a faulty mRNA and thus can still be detected with qPCR.
What should I do to confirm my knockout? Any suggestion will be appreciated.
Anh Hoang Le, your variants (may be not exhaustive) are:
1. Functional test for your protein ( if available)
2. NGS sequencing of KO area and confirmation that is 100% nonsense mutation(s) - usually out of frame deletion(s) or/and insertion(s). And! that it is early exon, but not too early if there is an alternative start site(s) for your gene.
3. Multiple TaqMan probes - especially targeting the 3' of you mRNA, as it should be most sensitive to nonsense degradation (check if this true, I am not 100% sure on that)
4. Order your gene antibody, - it is not that expensive this days, not sure about time-line here :)
Colleagues, more ideas?
HI Anh,
I see 2 options for you:
1- sequence your gene to find the stop codon created by indel. This will allow you to say that the protein is not being translated.
2- which i like better, if you don't have an antibody against your protein, you can always use Mass spec to show that the protein is gone from your cells. it's super easy.
Let me know how i can help more.
Thank you both for your suggestion. I actually did think about mass spec but its a bit complicated at my place to just use mass spec to check for your protein. Nevertheless, I think it is useful to know some other ways around the problem. I'll look for some antibody first then see what else can I do.
Thank you very much indeed
- Badges
- Science topic