E1, E2, biotinylated Ubiquitin and the buffer are from Enzolife sciences ubiquitination kit ( BML-UW9920-0001 ). I want to check whether my protein of interest, which is a E3 from rice, has ubiquitintion activity or not. For this I have E.coli BL-21 strains expressing recombinant E3 protein with MBP tag at the N-terminal and 6XHis at the C-terminal. But I was unsuccessful in purifying this recombinant protein (E3). I have tried using Amylose resin and Ni-NTA beads. In both cases, the protein is not binding to the column. So, I cannot purify it. Instead of purifying recombinant E3 from E.coli, if I use the lysate in the ubiquitination reaction, will it work? For western, I will probe the blot with antibody against E3 and antibody against biotinylated Ub. Will this work?
Hi Neha,
Are you sure your MBP tagged protein is in the soluble fraction after you sonicate your cell. Maybe all the protein is going into insoluble fraction and thats why you are not getting any protein from the supernatant (soluble fraction).the lysate thing will work to check whether your E3 ligase will undergo ubiquitination or not. but you wont be able to quantify your E3 ligase.
I centrifuge the suspension after sonication. I do see bands for my protein (E3 ligase) in supernatant and pellet after running them on SDS-PAGE. 50% of the protein seems to be in supernatant and the remaining in the pellet.
I want to check whether the protein has ubiquitination activity or not. So I guess the working with the lysate might help.
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Physical Phenomena
- Luminescence