In-vitro ubiquitylation assay with iped cullin complexes



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tarix26.02.2017
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#9722
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 b-ME, protease inhibitors

  • aliquot, freeze in -80 oC.

  • Ubcs can only be thawed once!

2. 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

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!))

----


20ul Total



  • Incubate 2 h at 30 oC

  • Run on 10% Gel.

  • Anti-Human-Ubiquitin blot

Comments:



  • 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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