A protocol for GST preparation and protein purification

[Arabidop]

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

[walid]

Hi
did u now which reagent could replace
DTT(dithiothreitol; Cleland’s reagent)
in protein extraction!...?