Hi,

The first question is «are you sure that your deletion worked ?». The second question is «are you sure that the specificity of both of your Ab is ok ? sometimes commercialized Abs don't really work». If yes, maybe that could be a question of protein isoforms. If you have more than one isoform (see on protein databases) maybe you deleted only one and in WB you don't see the protein, as your Ab recognizes only one isoform (that you deleted) of your denaturated protein. But by staining non-denatured proteins in whole cells used Ab recognize a common epitope for all isoforms ?

I bet it's an isoform issue as [email protected] you have an isotype control?

Hello Muhammad Uzair Rehman

I agree with Livia Odagiu regarding protein isoforms. You should try doing RT-PCR using primers spanning the specific exon-exon junction of every transcript and check that out by comparing it with wild-type.

Malcolm Nobre Livia Odagiu I have performed ShRNA based knockdown so there wouldn't be a guide RNA