- Transform UbcX pET28 plasmids into BL21 on kan/CAM plates
- Grow up 20 ml cultures in LB/kan to OD 0.6 and induce expression by adding 500 uM IPTG for 3.5 h at RT
- Lyse in 15 ml Falcon tubes in 3 ml IP-LB 0.2 % Triton X-100, 10 ul b–ME, protease inhibitors
- aliquot, freeze in -80 oC.
- Ubcs can only be thawed once!
- Cullin complexes
- grow 100 ml yeast Cullin-13xMyc Δcsn5 (cullins fully neddylated)
- lyse in 2ml IP-LB + proteinase inhibitors, wash beads again, final vol. ~5ml
- incubate 2hrs @ 4 oC with 60 ug 9E10
- add 300 ul protein A Sepharose (equilibrated in IP-LB), incubate 1h @ 4 oC
- wash 5x in IP-LB, inhibitors
- equilibrate in 1 x HEPES, pH 7.4
- Ub-Assay (per tube reaction)
10ul Cullin IP-beads, spin, take off SN
2ul UbcX bacterial lysate
1ul E1 (VS87 or commercial rabbit E1 from Boston Biochemicals)
2ul 10 x Human ubiquitin monomer (8 uM)
2ul 10 x Reaction Buffer (40 mM MgAc, 10 mM DTT, 1 mM PMSF)
2ul 10 x ATP regenerating system (20 mM HEPES pH 7.4, 10 mM ATP, 300 mM creatine phosphate, 10 mM MgAc, 1.5 mg/ml creatine kinase, 10% glycerol)
11ul 1 x HEPES (20 mM HEPES, pH 7.4, 100 mM KAc, 1 mM DTT (add fresh!))
- Incubate 2 h at 30 oC
- Run on 10% Gel.
- Anti-Human-Ubiquitin blot
- Not more than one freeze-thaw cycle of E2 containing protein lysates or 10x ATP-Buffer
- Aliquot and freeze all reagents
- Add DTT freshly to HEPES from stock sln.
- Add protease inhibitors fresh before start