Adapted from Pierce Glutathione Spin Column protocol

Bring column and sample (cytosol) to room temperature.
Prepare sample by mixing cytosol with Equilibration/Wash Buffer so the total volume equals at least 2mL. If volume is greater than the column (~8mL), steps 7 & 8 can be repeated until the entire sample has been processed. Do not exceed resin binding capacity (~200mg).
Remove the bottom tab (or rubber cap) from the Pierce Glutathione Spin Column by gently twisting. Place column into a centrifuge tube (15mL falcon tube).
Centrifuge to remove storage buffer (all centrifuge steps are 2 minutes @ 700 x g). Discard flow-through. Reuse centrifuge tube. (May require a second spin to remove all of the buffer.)
Equilibrate column with 2mL of Equilibration/Wash Buffer. Allow buffer to mix with resin.
Centrifuge to remove buffer. Discard flow-through. Reuse centrifuge tube.
Add the sample to the column. Incubate for 30-60 minutes at room temperature on a rocking platform to maximize GST binding to the resin.
Centrifuge. Discard flow-through. Start a new centrifuge tube.
Wash resin with 2mL of Equilibration/Wash Buffer. Centrifuge. Keep flow-through labeled as “Wash #1”. Start a new centrifuge tube. Repeat this step two more times collecting each fraction as “Wash #2” and “Wash #3”.
Measure the absorbance of the washes at 280nm (blank=Equilibrium/Wash Buffer). Absorbance of washes 1-3 should decrease to near zero (as non-GST proteins wash out of the column). If not, perform additional wash steps. After all washes, start a new centrifuge tube. Discard washes.
Elute GST proteins from the resin by adding 1mL of Elution Buffer. Centrifuge. Keep flow-through labeled as “Elute #1”. Start a new centrifuge tube. Repeat this step two more times collecting each fraction as “Elute #2” and “Elute #3”.
Measure the absorbance of the elutions at 280nm (blank=Elution Buffer). Absorbance of elutions 1-3 should decrease to near zero (as GST proteins wash out of the column). If not, perform additional elutions to get as much protein as possible.
Nanodrop to quantify protein in each elution (protein setting, Elution Buffer as blank).

Procedure for Glutathione Agarose Regeneration

The column may be used at least five times without affecting protein yield or purity.

Add 5mL of Regeneration Buffer #1. Centrifuge twice, discarding flow-through after each.
Add 5mL mq-water. Centrifuge twice, discarding flow-through after each.
Add 5mL of Regeneration Buffer #2. Centrifuge twice, discarding flow-through after each.
Add 5mL mq-water. Centrifuge twice, discarding flow-through after each.
Add 5mL of 0.05% sodium azide. Cap bottom and top of column. Mark column to show it has been used. Store at 4C.

RECIPES

Equilibration/Wash Buffer

50mM Tris, 150mM NaCl, pH 8.0.

Need at least 8mL/sample + 1mL for blank

For 1L:

6.1g Tris
8.8g NaCl
Adjust to pH 8.0 with HCl
Water to 1L

Elution Buffer

50mM Tris, 150mM NaCl, pH 8.0 containing 10mM reduced glutathione.

Need at least 3mL/sample + 1mL for blank

For 60mL:

184mg reduced glutathione
~60mL Equilibrium/Wash buffer
Adjust to pH 8.0 with NaOH
Equilibrium/Wash buffer to 60mL
Store at 4C

Regeneration Buffer #1

0.1M Tris containing 0.5M NaCl and 0.1% SDS, pH 8.5

Need 5mL/sample

For 500mL:

6.1g Tris
14.6g NaCl
0.5g SDS
Adjust to pH 8.5 with HCl
Water to 500mL

Regeneration Buffer #2

0.1M sodium acetate containing 0.5M NaCl and 0.1% SDS, pH 4.5

Need 5mL/sample

For 500mL:

4.1g sodium acetate
14.6g NaCl
0.5g SDS
Adjust to pH 4.5
Water to 500mL

Sodium azide

0.05% Sodium azide

Need 5mL/sample

CAUTION! Sodium azide powder is toxic and explodes in contact with metal. Place the scale in the hood on a large paper sheet to cover the metal surface. Cover the metal scale platform with paper and dispense into a plastic weigh boat. Pour from the container–no metal instruments. Mix by swirling–no magnetic stir rod. Store the leftover sodium azide powder in the lockbox with controlled substances.

For 1L:

500 mg Sodium azide
1L water.
Store at 4C