Glutathione Agarose

• High binding capacity >10 mg/mL.
• Delivered as a 50 % suspension
• Average agarose bead size: 40 μm

• Lysis Buffer
• Wash Buffer
• Elution Buffer
• Ice bath
• Refrigerated centrifuge for 50 mL tube (min 10,000 x g)
• 50 mL centrifuge tube
• Micropipettor and Micropipetting tips
• Disposable gravity-flow columns with capped bottom outlet, 2 ml
• 
  Optional: 15 mL conical propylene tubes pH meter
• End-over-end shaker
• UV/VIS Spectrophotometer
• SDS-PAGE buffers, reagents and equipment
  Optional: Western Blot reagents and equipment

Sample Volume for an assay: >200 mL E.coli culture volume or corresponding quantity. Protocol can be scaled up easily.

All buffers should be prepared fresh.

• Lysis buffer: Tris-HCl, pH 7.4, 125 mM, NaCl 150 mM, DTT 1 mM, EDTA 1 mM, Lysozyme 1 mg/mL, Triton X-100 1 % (v/v), Protease inhibitor 1x
• Wash buffer: Tris-HCl, pH 7.4, 125 mM, NaCl 150 mM, DTT 1 mM, EDTA 1 mM.
  Optional: ATP buffer: Tris HCl, pH 7.4, 50 mM, ATP 2 mM, MgSO4 10 mM
• Elution buffer: Tris base, pH 7.4, 125 mM, NaCl 150 mM, Triton X-100 0.1 % (v/v), Reduced glutathione 50 mM, DTT 1 mM
  Note: Optimal buffer conditions may vary depending on the protein of interest. Optimal pH can be varied from pH 7.4-8.0. Some proteins may require addition of protease inhibitor cocktail, EDTA, 1-5 mM DTT, 1 % BSA, or detergents such as 0.5-1 % Igepal CA-630 (Nonindet P-40) or 0.5-1 % Tween-20.

Protein purification protocol:

1. Thaw the E. coli cell pellets corresponding to 200 mL bacterial culture on ice for 15 min. Optional: Freezing the cell pellet at -20 °C for 30 min prior to incubation at room temperature improves lysis by lysozyme.
2. Resuspend the cell pellet in 10 mL Lysis Buffer and pour it into a 50 mL conical centrifuge tube. If the solution is very viscous, add 3 units Benzonase® per mL E.coli culture volume to the lysis buffer. Alternatively or additionally, sonicate the lysate to improve cell disruption.
3. Incubate on an end-over-end shaker at room temperature for 30 min, or at 4 °C for 1 h, depending on the temperature stability of the protein.
4. Centrifuge the lysate at 10.000 x g for 30 min at 2-8 °C and carefully collect the supernatant without touching the pellet. Note: The supernatant contains the cleared lysate fraction. We recommend to take aliquots of all fractions for SDS-PAGE analysis.
5. Resuspend the HighSpec Glutathione Agarose by inverting the bottle until the suspension is homogeneous. Transfer 1 mL of the 50 % suspension (corresponding to 500 μL bed volume) into a 15 mL conical centrifuge tube. Allow the resin to settle by gravity and remove the supernatant. Tip: Alternatively, resin equilibration can be performed directly in the disposable gravity flow column.
6. Add 5 mL of Wash Buffer and gently resupend the suspension to equilibrate the resin. Allow the resin to settle by gravity and remove the supernatant.
7. Add the cleared lysate prepared in step 4 and incubate at 4 °C for 1 h on an end-over-end shaker. Tip: Alternatively, batch binding can be performed directly in a gravity flow column with closed bottom and top outlets.
8. Pour the complete suspension into a disposable gravity flow column with a capped bottom outlet.
9. Remove the bottom cap of the column and collect the flow-through.
10. Wash twice with 2.5 mL each of Wash Buffer.
11. Optional: To remove contaminants such as chaperones, perform an additional wash step with ATP buffer.
12. Elute the GST-tagged protein by adding 0.5 mL Elution Buffer.
13. Repeat step 11 five times, for a total of six elutions. Collect each elution fraction separately. Optional: Incubate the resin for 15 min in Elution Buffer before collecting the eluate to increase protein yields.
14. Determine the protein concentration of the elution fractions with Bradford assay, using BSA as protein standard.
15. Analyze all fractions by SDS-PAGE. Note: Do not boil membrane proteins. Instead, incubate the sample at 46 °C for 30 min in preparation for SDS-PAGE analysis.
16. Optional: Perform Western Blot experiment using GST Antibody.

Analyze by SDS-PAGE, Bradford Assay or spectrophotometrically.