Not all antibodies can be used for knockout confirmation since some WB antibodies can bind to both WT and knockout equally (since the antibody recognizes nonmutated regions from the knockout). Thus please let me know if anyone has used a TLR2 antibody for genotype confirmation in TLR2-/- mice through western blot.
Hi Sreeram
When you say knockout, it means you no longer have TLR2 protein. So, the question of antibody binding to mutated or non-mutated doesn't even arise. On the other hand, if you are introducing a mutation within TLR2 gene, it is an issue of protein function. To assess if your genomic mutation worked, you need to perform a functional assay directed towards TLR2 activation and subsequent read-out (which can be cytokine release, cell activation, cell proliferation, or even differentiation).
In knockout testing, TLR2 protein expressed must be shorter than wild type protein while its amount is smaller than normal level. Abnormal TLR2 might be able to bind for antibody of this protein, depending upon the recognition of antibody. If TLR2 gene was mutated ato the positions of active site of protein, no protein should be expressed in any cells of whole body, resulting no immunoreaction to TLR2 antibody in western blotting method. Tateki Kikuchi
Hi,
Thanks for the responses.
V. Anaparti: Thanks for feedback. I'm afraid I cannot agree with you on the the argument that the question of "mutated/non mutated" protein doesn't arise in a knock out system, because the mutation in this case is only a substitution of the functional domains with neomycin resistance. Knockout here could just mean knocking out of the functional protein. As long as upstream promoter sites are not affected due to neoR substitution there is no reason for the protein to not be produced even if it is nonfunctional. And there is no reason to not have antibodies which cannot recognize both the proteins (functional vs nonfunctional forms). Hence, I was wondering if someone has a knockout validated antibody I could just use it to try. But like you said functional experiments must be done to check for read-out and thats already done for confirming the knockout phenotype via cytokine readouts. Knockout confimred using flow cyt as well. Thanks.
Tateki Kilkuchi: Thanks for feedback. yes, since there will be a molecular size difference between the WT and mt I can decipher it using standard PCR, and if lucky I could also find an antibody that may bind only to the WT.
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