Glutathione MagBeads

• High binding capacity >10 mg/mL.
• Delivered as a 25 % suspension
• Average magnetic agarose bead size: 25 μm

• Lysis Buffer
• Wash Buffer
• Elution Buffer
• Ice bath
• Refrigerated centrifuge (min 10,000xg)
• Micropipettor and Micropipetting tips
• 1.5 mL conical microcentrifuge tubes
• Magnetic holder for microcentrifuge tubes (for separation of magnetic beads)
•  pH meter
• Vortex
• End-over-end shaker
• SDS-PAGE buffers, reagents and equipment
  Optional: Western Blot reagents and equipment

Sample Volume for an assay: >10 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 pellet corresponding to 10 mL bacterial culture on ice. 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 1 mL Lysis Buffer. Add 30 U Benzonase® (3 units/mL bacterial culture) to the lysate to reduce viscosity caused by genomic DNA.
3. Incubate for 30 min on ice, if necessary for protein stability. Otherwise, incubating at room temperature (20-25 °C) may be more efficient.
4. Centrifuge the lysate for 30 min at 10,000xg and 2-8 °C. Collect the supernatant. 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 MagBeads by vortexing. Transfer 40 μL of the 25 % magnetic beads suspension into a conical microcentrifuge tube. Note: Depending on the protein expression rate, the quantity of magnetic bead suspension can be adjusted from 2-200 μL.
6. Add 500 μL Lysis Buffer to the Glutathione MagBeads and mix by vortexing. Place the tube on a magnetic microtube stand until the beads are separated and discard the supernatant.
7. Pipet 1 mL of the cleared lysate onto the equilibrated MagBeads, and incubate the mixture at 4 °C for 1 h on an end-over-end shaker.
8. Place the tube on the magnetic microtube stand until the beads separate and remove the supernatant.
9. Remove the tube from the magnet. Add 500 μL Wash Buffer and mix by vortexing. Place the tube again on the magnetic microtube stand and allow the beads to separate. Remove the supernatant.
10. Repeat step 10 twice. 12: Optional: To remove contaminants such as chaperones, perform an additional wash step with ATP buffer.
11. Elute the GST-tagged protein using 100 μL Elution buffer. Note: Depending on the protein expression rate and desired protein concentration, the elution volume can be adjusted from 25 to 500 μL.
12. Repeat step 12xxx Collect each elution fraction in a separate tube.
13. 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.
14. Optional: Perform Western Blot experiment using GST Antibody.

Analyze by SDS-PAGE, Bradford Assay or spectrophotometrically.