I am working on one gene having 19 exons with 4 transcripts. A deletion mutation (one bace pair deletion) is present in all the four transcripts of this gene. It leads to frameshift and make a stop codon after 6 amino acids. My question is that i have done reverse transcriptase pcr and found no splice variant. After that i did western blot and got the equal amount of protein like in control what could be the possibilities, principally there should not be any protein or if so then it should be significantly reduced?
Hi Maria,
Your assumption about the deletion mutations seems to be based on the database sequences. It may not be correct for all the four transcripts. There is likely to be some mistake in the annotation. Clone the cDNA and sequence it from both the samples...
best regards
Thank you Ajay, i want to tell you that i have sequenced cDNA: there is deletion in Mutant , so if there is deletion then it should not code any Protein and if so then it should be significantly reduced. also i did not find any splice variant.
If only one allele in the tissue you are analyzing is affected by the mutation the amount of protein on the WB might not change that much. You need to check for equal loading and do a semi-quantification of the protein.
Thank you sabine, but both of the allele are affected, and also this gene has no pseudogene.
Did you sequence from both sides and are you sure that you are not looking at a sequencing artifact? Can you upload the sequence chromatograms?
Hi
I also agree with Sabine above. Do not take the sequence output directly given by the instrument. Some times you may need to see carefully and edit the chromatogram. And also verify it in both the strands. How specific is your antibody...is it possible it can detec some other protein also?
best regards
Thank you Ajay and Sabine, i am also thinking for a new antibody. and also i must have to look at its sequence once again.
Maria
one possibility is that there is an inframe start codon.
Also check ExAc web site to check the frequency of your deletion .
Ram. S Puranam
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