poly ubiquity nation detection - (May/05/2014 )
I am trying to detect polyubiquitination in a cell lysate that was treated with the proteasome inhibitor bortezomib. I used the ordinary procedures for western blotting but I could not see a smear when I probe the pvdf membrane with antiubiquitin antibody. Is there something different in the procedures t that I should do to get that smear? Thanks a lot
you are trying to detect poly-ubiquitination in whole lysate, if so you can detect in direct western with more protein than usual. alternatively you can IP any known proteasome targets or total ubiquitin itself and western with anti-ubiquitin. if you still having problem on detecting endogenous ubiquitin, you can overexpress taged ubiqutin, treat with bortezomib and detect in direct western or IP.
May be you can also consider this (http://hamptonlab.ucsd.edu/Protocols/AutoclavingBlots.pdf) to increase the detection of ubiquitin immunoreactivity.
Finally I could detect polyubiquitinated proteins easily by loading double the usual amount of proteins and using a very good antibody from millipore fk1 clone