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In-vitro ubiquitylation assay with IPed cullin complexes

  1. E2s
  • 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 bME, protease inhibitors
  • aliquot, freeze in -80 oC.
  • Ubcs can only be thawed once!
  1. 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
  1. 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!))

—-

20ul    Total

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

Notes:

  • 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

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