I might be over thinking this but my problem lies with understanding how only a long protein isoform can be expressed when the short isoform is encoded by the same region, just 85 amino acids shorter. When I carry out a western blot using 4 different antibodies (2 of which can detect both isoforms) the only isoform to show up is the long isoform. A qPCR was then carried out using isoform 2 specific primers which showed no expression at all. Could somebody explain to me how this happens or possibly point me in the right direction?
Could you please elaborate the specifics, like, is this about a transgene or endogenous gene? two isoforms arise due to alternative splicing?
This is a novel endogenous human gene. Which is not well understood. I am unsure if the isoforms arise due to alternative splicing unfortunately but isoform two is transcribed using an alternative start codon further down the gene.(making it shorter)
The splicing factor that generates the shorter isoform may not be present. If it is not, then you would only see the larger isoform.
If the isoform2 is shown to express but if you cannot detect even the mRNA, my thinking is that it might express under certain specific conditions or in specific cell types.
Also I have another question, if these isoforms are due to alternative start codon, how did you design isoform2 specific primers?
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