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!...?

[jyothi83]

hi all,

I am working on cleavage of GST CypA fusion protein,cleavage is goodn and i futher proceeded to remoove GST by using GSH agarose beads,but after treating the cleaved protein to GSH AGAROSE BEADS ,the concentration of cypA drops down drastically very low.Kindly suggest me how to improve and conmcentrate the cypA