i created a huntington's disease model using plasmide gfp-q74 and gfp-q23(from addgene) in SHSY5Y cell line. then i made BCA for quantification of huntingtin protein and then i made western blotting (using ab109115 1'ab) for quantification of amount of huntingtin protein. but i met a problem following. Why is it smear in the membrane, is it normal? and why both q74 is different?
Hi Esra, protein smearing in SDS PAGE can be caused by posttranslational modifications. proteinase cleavage or both.
According to: Article The biological function of the Huntingtin protein and its re...
the protein has multiple sites that can lead to heterogeneity in SDS PAGE.
Dear Esra,
I am working a lot on huntingtin cleavage and I agree with Steingrimur that the lower "smear" underneath the full-length band may be proteolytic fragments or other PTMs of the over-expressed huntingtin protein. I could imagine that you could get a better resolution and a sharper appearance of those fragment bands if you had used a different WB protocol (e.g. different PA gel).
Regarding the "difference" between your both Q74 lines, I'd guessed that the first line is just expressing a Q23 huntingtin instead of the Q74 variant, as it looks really comparable in size and appearance to Q23 line in the fourth lane. Furthermore, your EGFP empty vector looks more like a Q74 huntingtin. Was there a mix-up? Have you additionally detected this WB with an EGFP antibody?
Best,
Jonasz
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