In-vitro ubiquitylation assay with iped cullin complexes

In-vitro ubiquitylation assay with IPed cullin complexes
1. E2s

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  Transform UbcX pET28 plasmids into BL21 on kan/CAM plates
  
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  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
  
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  Lyse in 15 ml Falcon tubes in 3 ml IP-LB 0.2 % Triton X-100, 10 ul b-ME, protease inhibitors
  
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  aliquot, freeze in -80 oC.
  
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  Ubcs can only be thawed once!
  

2. Cullin complexes

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  grow 100 ml yeast Cullin-13xMyc Δcsn5 (cullins fully neddylated)
  
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  lyse in 2ml IP-LB + proteinase inhibitors, wash beads again, final vol. ~5ml
  
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  incubate 2hrs @ 4 oC with 60 ug 9E10
  
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  add 300 ul protein A Sepharose (equilibrated in IP-LB), incubate 1h @ 4 oC
  
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  wash 5x in IP-LB, inhibitors
  
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  equilibrate in 1 x HEPES, pH 7.4
  

3. 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!))

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20ul Total

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  Incubate 2 h at 30 oC
  
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  Run on 10% Gel.
  
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  Anti-Human-Ubiquitin blot
  

Comments:

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  Not more than one freeze-thaw cycle of E2 containing protein lysates or 10x ATP-Buffer
  
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  Aliquot and freeze all reagents
  
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  Add DTT freshly to HEPES from stock sln.
  
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  Add protease inhibitors fresh before start