Yeast Stress in Media to increase ubiqutin conjugates - (Jan/17/2007 )
Hi, i need reliable possibility to induce ubiquitin conjugates levels for a Tap-Tagged Protein purification. I was not able to stress my cells the properly enough (polyubiquitin-blot). Even in double stress condition for 1h with 5mM DTT and 37°. In those cases i see only good in double deletion mutants (Rad23 und Dsk2).
Another possibility is to use aminoacid analoga (canavanine, AZC) which induce folding stress and ERAD. My question is how i have to handle it in this cases in media? I need this in media because i want to purify normal expressed proteins!
Im very happy for every small hint, thank you
Strange that DTT gave little effect. Maybe you can try tunicamycin or thapsigargin. I don't know the references by heart, but search on UPR+yeast+tunicamycin must give some results (a publication by K. Travers).