2 replies to this topic

[monica]

WHAT IS THE SIMPLE ASSAY FOR UBIQUITIN PROTEIN?

[sean]

what do want to assay? and is that ubiquitinated protein or the actual Ub itself?

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.

[Lex]

Guess, the point was about ubiq. assay. If so, you may follow this protocol.
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)
enjoy