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COUPLING GST/UBA PROTEIN TO GLUTATHIONE RESIN

Author: Stefanie Duttler, updated date: , view: 6, Q&A: 0
Tags: Ubiquitin and GST

Procedure


  • Resin is at 4deg, shake bottle well to homogenize slurry

  • Add 1ml of slurry to a 2ml tube

  • Spin at 2000 g 1-2 min

  • Wash 1ml of lysis buffer 3x

  • on last wash, remove buffer and add 1ml of purified protein

  • Rotate at 4°C for 1hr

  • Spin at 2000 g 1 min at room temperature

  • Remove supernatant and refreeze (safe to thaw 2-3 times)

  • Wash 1 ml of lysis buffer 3x

  • Resuspend in 1ml of wash buffer

  • Store beads at 4 deg, they are good for 2-3 months



Lysis Buffer

20mM HEPES  pH7.5

10mM MgCl2

50mM KCl

1% Triton X-100


Add before use, final concentrations:

100ug/ml Cyclohexamide

20mM N-ethyl maleimide

1X Protease Inhibitor cocktail

1mM PMSF

1X Superase-in (Ambion)


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