A classmate of mine tested nicastrin knockout and wildtype cells against antibodies, but never tested to make sure that the knockout cells did not indeed contain nicastrin. So far, I have performed immunoblots as well as preparing for PCR genotyping. Are there any other methods in which I can ensure that nicastrin is knocked out in the KO cells?
you can look at three level: DNA (PCR), RNA (RT PCR) and protein (Western blot)
you can look at three level: DNA (PCR), RNA (RT PCR) and protein (Western blot)
most precise way is to make western blott
Also try to resore it by over expression
Immunoblotting is quite reliable to confirm your KO.
If it is something more visual what you want, try immunohistochemistry and you can get a good image of the level of NCT in the cytoplasm of the cells after KO.
http://www.rndsystems.com/product_results.aspx?m=4198
But definitely, expression level (mRNA) is a must.
First you have to know how exactly this KO was created. Sometimes the protein is transcribed and translated but is non-functional, in which case you can detect the protein by WB (depending on your antibody). In other cases there is an insert in the gene that generates and early stop codon, or a Nemycin casette inserted in c-terminous,.. Depending on the method used for knocking out this protein you have to choose one or another method: DNA, mRNA or WB, and choose correctly the antibody or probe.
it depends what do you mean as KO and how you did this. A KO mouse (or generally speaking an organism) is usually KO by interruption of a gene through a cassette insertion (as. puromicin), and you usually do a PCR to genotype it by using a primer amplifying the cassette or the cassette plus part of the gene. If you want to be sure this KO doesn't express your protein you should first check that you impaired the production of the mRNA (rt-QPCR) and the protein as well (western, but also facs, ELISA on tissues or ELISA on supernatant, immunostaining, immunohistochemistry are all valid tools according to the protein), but this information is usually provided by the company/consortium that did the KO. The fact with KO models is that you need to know where your protein is expressed (tissue), the DNA is the same in every cells whereas the mRNA e protein profiles are different from one tissue to another.
In case you have KOdown a protein in a cell line with an shRNA (it means that it is transient) or KO (stable KO) your protein of interest by talents/zinc fingers or CRISPR you check first the plasmid insertion by PCR then the mRNA levels and the protein by FACS or immunostaining or western blott or ELISA (but only if the protein is released or on the cell surface). You always have to check the DNA >KO efficiency by mRNA and protein levels.
what would you do if the protein you knocked out doesnt have good specific antibody?
That's a tricky one.
I think you should check the mRNA levels. If you've got the sequence, you can make primers and run a PCR. Then if you don't find its messenger, I don't see why you should go and look for the protein.
I can think that if you've got a low specificity antibody you can still separate a fraction containing part of that protein. Then you'd need to characterise using more specific methods like mass spec.
Hope this helps
Javier Arturo Sánchez López , How do you check the mRNA level by qPCR method? Do you just extract mRNA from KO cells and WT cells and compare or you do any treatment to see the difference in induction of the targeted gene in KO and WT cells. It would be great help if you could share your methodologies.
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