Hi,

you can co-transfect small amounts of tagged ubiquitin (such as FLAG-Ub) and also add NEM inhibitor in lysis buffer to prevent deubiquitination (by cellular DUBs). FLAG-Ub will enable all types of lysine-dependent ubiquitination.

You can either do IP-HA under denaturing conditions and blot for FLAG (and HA) or do IP FLAG under denaturing conditions and blot for HA.

You can use unconjugatable (lacking terminal Gly) ubiquitin as negative control.

Good luck!

Hi Dr. Husnjak,

Thank you very much for your comments and suggestions. Well, I thought to overexpress Ubiquitin under strong promoter in my strain, but my PI did not like this idea. Therefore, I tried denaturing HA IP with denaturing buffer (10mM Tris-HCl, pH 7.4, 150mM NaCl, 0.5mM EDTA, 0.5 % Triton, 20mM N-ethyl-maleimide (NEM), and 1× protease inhibitor cocktail). I could see an enrichment in my bait. But my -ve control still gives Ub signal, so I guess I need to remove the background completely with high salt/detergent amount (using wash buffer with 250mM NaCl and 1.0% Triton) that might help. Would this condition be feasible to try? 

Hi,

it is enough to transfect 0.1 ug of tagged ubiquitin per 6well plate, or even less. Only a small fraction of your protein is most likely modified and it is sometimes not easy to detect it.

As far as your buffer is concerned, it is not denaturing enough, so you see residual ubiquitin binding in your negative control. Check our Nature Methods paper (2017) for HA agarose IP conditions. It will surely help to add more salt and detergent in washing steps, no doubt.

Furthermore, I do not know which type of ubiquitination you are after. If it is degradative ubiquitination (i.e. mainly K48), it might help to add MG132 to cells several hours before lysing, otherwise no.

Take care!

Hi Dr. Husnjak,

Thank you very much for your comments again. I will look for your 2017 Nature Method paper for this issue.