Posted 13 March 2002 - 06:13 PM
if u wanna visualize them, you can do a western blot with anti-Ub. if a protein is ub'd then it should glow. strips and reprobe with with antibody to the proteins that's ub'd.
if you want to assay for ubiquitinatin enzyme thats a entirely different story.
Posted 30 April 2002 - 10:13 AM
Let's say you wanna see Rad6 and you have antibody to it.
make an IP
wash your pellets twice with IP buffer and once in your ubiq.buffer (50mM Tris, pH7.5; 50mM KCl; 5mM MgCl2; 100ug/ml BSA)
spinn the pellets and remove "super"
put ATP (2mM), ubiquitin (2.6ug), histone H2B and enzyme (for instance, if you are using Rad6, you need E2-conjugating enzyme, amount depends on activity),
incubate 15 min at 30C
put loading SDS buffer and run your SDS PAGE (pref. gradient)
Make Western and probe with anti-ubiquitine antibody (try not to use the same species as for IP)