| 1A(ii) |
No polyubiquitinated product or free polyubiquitin chains after in vitro assembly |
Old stock of E1, E2, E3 enzymes |
Express and purify new enzyme stocks (or purchase commercially) |
| Incubation time too short |
Perform a longer time course |
| Ubiquitin input concentration too low |
Adjust the amount of monoubiquitin present in the assembly reaction |
| 1B(xi) |
Little or no immunoprecipitated ubiquitinated substrate detected |
Not enough cells used for pull-down |
Increase number of dishes |
| Inefficient pull-down |
Optimize pull-down conditions with fresh antibody/affinity gel |
| 5 |
No ubiquitin chain hydrolysis detected on SDS-PAGE gel |
Old stock of DUB |
Express and purify new stocks of DUB |
| Enzyme concentration too low |
Optimize enzyme concentration; can try several in parallel |
| Incubation time too short |
Increase incubation time, perform a longer initial time course |
| Substrate concentration too high |
Dilute substrate and repeat, can try several concentrations in parallel |
|
Box 1, step16 |
No DUB can be detected on SDS-PAGE gels |
Poor protein expression |
Use fresh aliquot of competent bacterial cells, optimize expression conditions (IPTG concentration, induction times and growth temperature). |
| No protein present in sample after elution from beads |
Old aliquots of Prescission protease |
Replace PreScission protease batch with a fresh one |
| Inefficient cleavage |
Optimize cleavage conditions (incubation time and buffer composition) |
| Eluted protein is not pure |
Insufficient washing of beads |
Wash with 2 L of GST buffer 2 and GST buffer 1 |
| Used too much Glutathione Sepharose 4B resin |
Optimize the amount of resin used |
|
Box 2, step 5 |
No ubiquitin chain hydrolysis detected on SDS-PAGE gel |
Old stock of DUB |
Express and purify new stocks of DUB |
| Enzyme concentration too low |
Optimize enzyme concentration; can try several in parallel |
| Incubation time too short |
Increase incubation time, perform a longer initial time course |
