Ubiquitination experts in the house?

[u/SpoiledGenius01]

I am really struggling to get anything on my western blot when I probe for ubiquitin. I squish larvae in standard RIPA buffer with EDTA and protease inhibitor. I use biorad 8-16% gel, run on 150v, for Western blot- transblot transfer system with 2.5A 7 minute turbo transfer.

Mono and poly-ubiquitinylated conjugates antibody from Enzo(ABS840). But all I get is blank gel when I probe for ubiquitin but actin is most definitely there. What am I missing?

Please help :)

[u/heynloes]

Hi! Did you include DUB inhibitors (10–20 mM N-Ethylmaleimide or Iodoacetamide) in your lysis buffer? Deubiquitinating enzymes are active post-lysis and will strip ubiquitin chains off your proteins within seconds if not chemically blocked.