We tried three different piezo-1 Ab's for western blot and we observe four bands 1. 250KDa+, 2. 100Kda, 70 KDa and 30 KDa. PIEZO-1 full length protein should only gibe 250+ KDa band. I would like to ask if other people also have similar issues and if anyone know that the bands have been described earlier as piezo-1 splice variants.
Dear Kapish Gupta,
no, there are no additional splice variants (which means mRNA splice variants) known for PIEZO-1. Moreover, there is no information for protein isofoms (potentially generated from mRNA variants) indicating that you should expect different bands, showing different isoforms.
It is more likely, that you possibly detect degradation/aggregation products of your protein (depending on isolation protocol (different lysis buffers lead to different band numbers and results!), storage temperature and duration etc.).
Moreover, have you ever testet the specificity of your antibodies by using recombinant proteins (not blocking peptides) to validate your Ab with showing a clear singe band in the correct range?
In my opinion, you should opimize your methods/protocoll or the antibodies used here are possibly of low quality.
Andreas Eisenreich thanks for you comment. I am using this antibody. https://www.novusbio.com/products/piezo1-antibody_nbp1-78537. on the website also it shows similar 4 bands. I think i need to change the protocol for isolation as piezo can be degraded. Can you suggest some good protocol is you have used it before?
Thanks
No sorry, I can't. But you should try different protocolls (esp. lysis buffers, e.g. RIPA, MCL etc.) and choose the best one for your further experiments.
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