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Purification of GST-tagged Proteins by Batch

 

Grow up at least 1L of bacteria expressing your protein of interest, harvest and pellet.  Store pellet in 50 mL falcon  (conical) tube at -80C until ready for this prep.

 

Thaw frozen pellets. 

Resuspend pellets in approximately 5 volumes (usually 20-25mL) of:

1X PBS

5 mM EGTA

2 mM  PMSF

2 mM Benzamidine Hydrochloride

200 ug/mL Lysozyme

15 mM DTT

0.25 M KCl

 

On ice, dounce homogenize pellet until fully resuspended.  Transfer back into 50 mL conical tube.

 

Using 150 mL stainless steel beaker, sonicate suspension to shear DNA, 50% duty cycle, output between 2 and 3.  Cool samples between sonications.   Sonicate until no longer goopy.  Take 100 uL sample, spin down at max speed in microcentrifuge, aspirate off supernatant.  This is the insoluble pellet sample for the gel.

 

Transfer rest of sonicate into an oak ridge tube and spin in SS-34 rotor at 18,000 rpm for 50 minutes at 4C to clarify the sonication supernatant.  Save the pellet for insoluble material.  Take 100 uL of the supernatant for gel.

 

At this point, many people like to do another high-speed spin in the ultracentrifuge using the 50.2 Ti rotor at 35,000 rpm for another 50 minutes.  I have found that this is not essential.

 

While the spins are going on, prepare the GST-agarose (in the 4C refridgerator).  Aliquot 2 mL of the slurry into 50 mL conical tubes (wash out tubes that had frozen pellets in them and re-use).  Wash beads 2X in PBS. 

Then, wash into the resuspension buffer used above to equilibrate the beads to the sample.

 

Pour the clarified supernatant onto the beads, and incubate rotating end over end at 4C for several hours to overnight (overnight maximizes binding) to allow protein binding to the GST-agarose.

 

Spin the protein bound beads at 3200 rpm in the tabletop clinical centrifuge for 5 min, preferably at 4C.  Carefully remove the supernatant, and save in a new conical tube.  Take 100 uL of this 'flow through' for gel.

 

Wash the beads 3X in 1X PBS/300 mM NaCl/0.1% TX-100/1 mM DTT.

Wash the beads 2X in 1X PBS/300 mM NaCl/1 mM DTT.

 

Remove all but approximately one mL of buffer from beads.  Transfer beads to a 2 mL eppendorf.  Spin down at 4000 rpm at 4 C.  Remove as much buffer as possible without losing any beads.

 

Elute protein from beads, 1 mL at a time with:

50 mM Tris-HCl, pH 8.0

1 mM DTT

0.25M KCl

5 mM GST

 

Monitor protein concentration with Bradford assay.  Run samples on SDS-PAGE to assess purity.

 

Pool cleanest and most concentrated fractions.  Protein can be frozen down in liquid N2 and stored at -80C until ready to process further.

 

 

This page last modified 08/11/2011

The Stukenberg Lab and the Burke Lab are in the Department of Biochemistry and Molecular Genetics at the University of Virginia