I am interested in ubiquitin western blotting.
I would like to know, if I use ubiquitin monoclonal antibody, will it give only a single band corresponding to it or will it be able to recognize other bands (where ubiquitin is covalently attached to other proteins) and give a ubiquitin ladder?
Kindly provide me with your precious advice.
You can start with P4D1, what would give you the polyubiqutinated protein smear/ladder looking for on your western, and if you want to distinguish from poly and mono ubiquitination you can use FK1 or FK2 and compare with P4D1. Some batch of P4D1 can give you very high background, use BSA and increase washing steps would help.
Hi Pooja
I think if you want to see covalent-attached ubiquitin for the total cell proteins or for protein of interest, you may need harsh lysis buffer as to remove any non-covalent (hydrophobic) interaction of ubiquitin with another proteins ......thus you also decrease the "noise" in western blot....
i attach a protocol it will help you
BEST
The P4D1 antibody from Santa Cruz Biotechnology has given me nice and clean results. It is better if you use PVDF instead of nitrocellulose and denature the membrane before blocking. Good luck!
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