DAY 1 - Resin Preparation

1. Hydrate the lyophilized powder of glutathione agarose (Sigma prod.# G4510, claimed binding capacity = 5-10mg protein/mL resin, one gram powder swells to about 14mL gel) in deionized water at 200mL/g. 90-95% swelling occurs within about 30 minutes at room temp, but it is ideal to soak the resin overnight at 4^oC for complete swelling.

DAY 2 - Binding and Thrombin Digest

1. Prepare 30mL PBS buffer¹, pH7.4 (add additive, such as 0.5M Urea or 0.2% NP40, if necessary).

2. Weight 5-10g cell pellet. Add 50mL lysis buffer, plus 125ul 0.4M PMSF, 10ul 10mg/mL DNase, and 50mg lysozyme. Resuspend cell in lysis buffer well.

3. French press twice at 1,000 psi.

4. Centrifuge lysate at 15K rpm for 30 minutes.

5. Prepare 1-2mL (depending on expression level) glutathione column. Wash with 10 column volumes of lysis buffer (without enzymes).

6. Load the lysate supernatant on the GSH column. For better binding, resuspend the resin using the lysate supernatant and nutate at 4^oC for an hour, then load the lysate on the column 4 times.

7. Wash column twice with 10 column volumes of wash buffer².

8. Wash column with 10 column volumes of Thrombin digest buffer³.

9. Add about 3mL Thrombin digest buffer containing 100 units thrombin.

10. Close column, leave at room temp overnight.

DAY 3 - Elution

1. Collect 0.5-1mL fractions (about 20 fractions) carefully with washing buffer.

2. Wash with about 5mL of elution buffer ⁴.

3. Check OD at 280nm and run SDS gels for fractions.

Solution Preparation (Note: filter the solutions with 0.2-micron syring filter & store at 4^oC.)

1. PBS buffer
Use Sigma phosphate buffered saline tablet (prod.# P-4417)
Dissolve 1 tablet in 200mL deionized water at 25^oC to obtain:
10mM phosphate buffer, pH 7.4
2.7mM KCl
137mM NaCl
Additive if necessary:
      (e.g. 100ul NP40 in 50mL for making 0.2% NP40)
      (e.g. 1.5g Urea in 50mL for making 0.5M Urea)

2. Wash buffer
50mM Tris, pH 7.5 (2.5mL 1M Tris, pH 7.5 in 50mL)
150mM NaCl (7.5mL 1M NaCl in 50mL)
Additive if necessary:
      (e.g. 100ul NP40 in 50mL for making 0.2% NP40)
      (e.g. 1.5g Urea in 50mL for making 0.5M Urea)

3. Thrombin digest buffer
50mM Tris, pH7.5 (2.5mL 1M Tris, pH7.5 in 50mL)
150mM NaCl (7.5mL 1M NaCl in 50mL)
2.5mM CaCl₂ (125ul 1M CaCl₂ in 50mL)
Additive if necessary:
      (e.g. 100ul NP40 in 50mL for making 0.2% NP40)
      (e.g. 1.5g Urea in 50mL for making 0.5M Urea)

4. Elution buffer
50mM Tris, pH7.5 (2.5mL 1M Tris, pH7.5 in 50mL)
150mM NaCl (7.5mL 1M NaCl in 50mL)
5mM glutathione (0.077g anhydrous reduced glutathione, MW=307.3, in 50mL)
Additive if necessary:
      (e.g. 100ul NP40 in 50mL for making 0.2% NP40)
      (e.g. 1.5g Urea in 50mL for making 0.5M Urea)

Sigma Product Information Sheet for Glutathione Agarose

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