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A protocol for GST preparation and protein purification
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| A protocol for GST preparation and protein purification First defrost your cells. Add benzamidine to final conc 5 mM Add PMSF to final conc 1 mM -lyse cells 2 or 3 times in french press -spin lysed cells (15k, 120 min @ 4C in ss34 rotor) -swiftly remove supernatant and keep on ice -take 2.66666666 ml GS-4B bead slurry per litre of bacterial culture -wash GS-4B beads 3 times in 10x volumes of TBS buffer -spin down beads @ 1600 rpm, 10 min and discard supernatant -resuspend in fresh buffer -take up beads in same volume of TBS buffer to give 50% slurry -add 2mL of 50% GS-4B bead slurry in TBS per litre of culture -put on roller @ 4C for 45 min to bind -spin down beads & bound protein (1600 rpm, 10 min) -wash beads 3x in 10x volumes 20 mM tris, pH 7.5, 50 mM NaCl, 3 mM DTT, 0.02% azide -incubate for 15 min on roller in each wahsing step -spin down beads & bound protein (1600 rpm, 10 min) -keep supernatant of each wash to test for protein -take up beads in equal volume of buffer -add CaCl2 to 10 mM -cleave with 25 units thrombin per litre culture (thrombin requires 10 mM CaCl2) -cleave for 2hrs @ RT, then overnight @ 4C with rolling -take sample from supernatant -check all samples on gel -if cleavage good, spin beads @ 1600 rpm for 10 min -wash beads 1-2x in equal volume of buffer -pool supernatants for gel filtration -if the cleavage is poor: remove GST from beads with 15mM glutathione & re-cleave |
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