I am trying to check the ubiquitination status of a protein by transfection of HA tagged ubiquitin followed by IP (for my protein) which in turn is followed by 24 hours serum starvation, before MG132 treatment. I use 1mg of protein conc for the IP and run 100ug of the lysate as input. Previously I used to get a very thick smear in the inputs that correspond to the HA-Ub transfected cells (correct result). Of late I am not seeing any bands in my inputs - I see the bands on ponceau staining but nothing after probing with anti-HA. I have tried all kinds of things to trouble shoot and now I have no idea what else to try.
Things I have already tried:
change HA-Ub plasmid stock (I checked if it worked by just transfecting it into cells and directly checked for expression of HA-Ub, it worked but when I use the same for my main experiment it goes kaput)
change lysis buffer
changed cell line
transfection reagent
sonication of lysates
used fresh Antibody stock for probing.
HA-ubiquitin is a common approach to monitor ubiquitination, with a 'few string attached'. You might want to try to pull down your protein using 'ubiquitin traps', TUBEs (LifeSensors). No need to express HA-Ubi!
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