体外泛素化标准流程.doc

In vitro ubiquitination assay A. Total 15ul Reaction setup Rxn-mix (ul) E1-mix (ul) 5x Buffer 2.0 1 ATP (100mM) 0.5 - Ub (10mg/ml) 1.0 - UbcH7 (~10mg/ml) 0.5 - E1 - 0.5 E3 Sf1 (5ul) Sf2 (3ul) 5.0 - Compounds (5-10uM) + (final DMSO can be up to 20% for Smurfs) dH2O adjust to total 15ul B. Mix reaction-mix by vortex briefly, spin down and aliquot appropriate amount base on calculation into 1.5ml eppendorf tube containing Smurfs. Mix reaction by tapping. Add equivalent amount of compounds and mix. C. Pipette E1 into 5x Buffer in a separate labeled tube (E1-mix), mix properly and spin down. Aliquot E1 to the lid of the 1.5ml tube and close gently. Spin all tubes at the same time and incubate at room temperature (lab bench) for 1 hour. Terminate reaction by adding sample buffer. Either store away immediately at –20C or boil for 4 min at 95C. Resolve on 7.5% SDSand incubate in Anti-Ub antibody (1:2000). 1x Buffer 50mM Tris-Cl pH7.5 2mM ATP (supplement to 5mM before the reaction is initiated) 10mM MgCl2 50uM DTT Stock 5 X buffer 250 mM Tris-Cl pH7.5 10mM ATP 50mM MgCl2 250?M DTT