• Wear protective gloves, clothing, eye, and face protection. Wash hands thoroughly after handling.
• Avoid freeze/thaw cycles and centrifugation which could damage the beads.
• Vortex samples for about 10 seconds before adding magnetic beads.
• Vortex beads for about 10 seconds and mix them well with lysates containing GST-tag proteins to ensure best performance.
• Elute GST-tag proteins from the beads completely.

• Si-Mag magnet rack
• Binding/Wash Buffer: PBS buffer (137mM NaCl, 2.7mM KCl, 10mM Na₂HPO₄, 1.8mM KH₂PO₄, pH7.4)
• Elution Buffer: 50mM Tris-HCl, 10mM Glutathione (reduced). Freshly made solution is recommended.

Protocol

LARGE-SCALE PURIFICATION

1. Sample preparation. Collect cells from E. Coli, yeast or other cell cultures (~100 mL). Re-suspend these cells with 10 mL of Binding/Wash buffer containing protease inhibitors (such as 1mM phenylmethylsulfonyl fluoride (PMSF). Lyse cells either by sonication or by using French press. Centrifuge the crude lysate at 14,000RPM for 15 min at 4°C.
2. Re-suspend and pipet out 5mL of GSH magnetic beads, wash them twice with water using Si-Mag magnet rack (or similar magnet racks made by other vendors).
3. Add GSH magnetic beads to cell lysates, incubate at 4°C or room temperature for 30 minutes. Addition of 1-2 mM DTT could increase the binding between GST-fusion protein and beads. Insert the tube into Si-Mag magnet rack for 30-60 seconds.
4. Remove supernatant by holding the magnet rack upside down or by pipetting.
5. Wash the beads with 5 mL of Binding/Wash Buffer. Make sure the beads get completely resuspended by vortexing. To reduce non-specific protein binding, it is recommended to add 0.1% Tween 20 to the Binding/Wash Buffer.
6. Repeat step 5 three times.
7. Elute proteins bound to the beads by adding 2-4 mL of Elution Buffer and incubating  5 minutes at 4°C or room temperature. Make sure that beads get completely resuspended by vortexing.
8. Immobilize beads by placing tube into a magnet rack for 30-60 seconds and transfer  protein solution into a clean tube.
9. Store purified protein at -20°C for a long-term storage.

SMALL-SCALE PURIFICATION

1. Sample preparation. Collect cells from E. Coli, yeast or other cell cultures (~10 mL). Re-suspend these cells with 1.5 mL of Binding/Wash Buffer containing protease inhibitors (such as 1mM phenylmethylsulfonyl fluoride (PMSF). Lyse cells either by sonication or by using French press. Centrifuge the crude lysate at 14,000RPM for 15 min at 4°C.
2. Re-suspend and pipet out 0.5 mL of GSH magnetic beads, wash them twice with water using Si-Mag magnet rack (or similar magnet racks made by other vendors).
3. Add GSH magnetic beads to cell lysates, incubate at 4°C or room temperature for 30 minutes. Addition of 1-2 mM DTT could increase the binding between GST-fusion protein and beads. Insert the tube into Si-Mag magnet rack for 30-60 seconds.
4. Remove supernatant by holding the magnet rack upside down or by pipetting.
5. Wash the beads with 1.5 mL of Binding/Wash Buffer. Make sure the beads get completely resuspended by vortexing. To reduce non-specific protein binding, it is recommended to add 0.1% Tween 20 to the Binding/Wash Buffer.
6. Repeat step 5 three times.
7. Elute proteins bound to the beads by adding 300-500 mL of Elution Buffer and incubating  5 minutes at 4°C or room temperature. Make sure that beads get completely resuspended by vortexing.
8. Immobilize beads by placing tube into a magnet rack for 30-60 seconds and transfer  protein solution into a clean tube.
9. Store purified protein at -20°C for a long-term storage.

Regeneration of IDA-GST Magnetic beads: