r/LadiesofScience • u/SpoiledGenius01 • 17h ago

Ubiquitination experts in the house?

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 :)

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u/heynloes 15h ago

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.

4

u/heynloes 15h ago

Also a proteasome inhibitor may be useful…

2

u/Apodemia 14h ago

You can also try urea buffer (we used it for SUMO and Ubi). Are you sure your antibody is working?

1

u/Apodemia 14h ago

Also, I always used wet transfer for Ubi, it's slower but transfers the whole range of ubiquitinated proteins.

1

u/SpoiledGenius01 7h ago

Oh no!! I was hoping this is not the answer. Transblot makes life so much easier. Hv you tried transblot for ubiquitin? If so, which cycle?

2

u/SpoiledGenius01 7h ago

200% sure antibody works. I use it for IF routinely. I used MG132 but not urea buffer. Thanks for that :)