Characteristics of Glutathione Sepharose® Products
Glutathione Sepharose® High Performance is recommended for high-resolution purification of GST-tagged proteins, providing sharp peaks and concentrated eluent. Glutathione Sepharose® Fast Flow is excellent for scaling up. Glutathione Sepharose® 4B has high capacity and is recommended for packing small columns and other formats including batch purifications.
Table A2.1 summarizes key characteristics of these three Glutathione Sepharose® chromatography media, and Tables A2.2 to A2.6 summarize the characteristics of the same media prepacked in columns and as 96-well plates. For more information, refer to Chapter 5 (Purification using Glutathione Sepharose® High Performance, Glutathione Sepharose® 4 Fast Flow, and Glutathione Sepharose® 4B).
Characteristics | Glutathione Sepharose® High Performance | Glutathione Sepharose® 4 Fast Flow | Glutathione Sepharose® 4B |
---|---|---|---|
Matrix | Highly cross-linked 6% agarose | Highly cross-linked 4% agarose | 4% agarose |
Average particle size | 34 µm | 90 µm | 90 µm |
Ligand concentration | 1.5–3.5 mg glutathione/mL medium (based on Gly) | 120–320 µmol glutathione/mL medium | 200–400 µmol glutathione/g washed and dried medium |
Binding capacity1 | 7 mg recombinant glutathione S-transferase/mL medium | 10 mg recombinant glutathione S-transferase/mL medium | 25 mg horse liver GST/mL medium |
Recommended flow velocity2 | < 150 cm/h | 50–300 cm/h | < 75 cm/h |
Chemical stability | Stable to all commonly used aqueous buffers, e.g. 1 M acetate, pH 4.0 and 6 M Gua-HCl for 1 h at room temperature | Stable to all commonly used aqueous buffers, e.g. 1 M acetate, pH 4.0, and 6 M Gua-HCl for 1 h at room temperature | Stable to all commonly used aqueous buffers. Exposure to 0.1 M NaOH, 70% ethanol, or 6 M Gua-HCl for 2 h at room temperature or to 1% (w/v) SDS for 14 d causes no significant loss of activity. |
pH stability | 3–12 | 3–12 | 4–13 |
Storage temperature | 4 °C to 30 °C | 4 °C to 30 °C | 4 °C to 8 °C |
Storage buffer | 20% ethanol | 20% ethanol | 20% ethanol |
1 The binding of GST-tagged proteins depends on size, conformation, and concentration of the protein in the sample loaded. Binding of GST to glutathione is also flow dependent, and lower flow rates often increase the binding capacity. This is important during sample loading. Protein characteristics, pH, and temperature, but also the media used may affect the binding capacity.
2 H2O at room temperature.
Chromatography media | GST MultiTrap 4B: Glutathione Sepharose® 4B |
Filter plate size1 | 127.8 × 85.5 × 30.6 mm |
Filter plate material | Polypropylene and polyethylene |
Binding capacity | GST MultiTrap 4B: Up to 0.5 mg GST-tagged protein/well |
Reproducibility between wells2 | +/- 10% |
Volume packed medium/well | 50 µL (500 µL of 10% slurry) |
Number of wells | 96 |
Centrifugation speed: | Depends on sample pretreatment and sample properties |
recommended | 100–500 × g |
maximum | 700 × g |
Vacuum pressure: | Depends on sample pretreatment and sample properties |
recommended | -0.1 to -0.3 bar |
maximum | -0.5 bar |
pH stability | Glutathione Sepharose® 4B: 4–13 |
Storage | 20% ethanol |
Storage temperature | 4 °C to 8 °C |
1 According to ANSI/SBS 1-2004, 3-2004, and 4-2004 standards (ANSI = American National Standards Institute and SBS = Society for Biomolecular Screening).
2 The amount of eluted target proteins/well does not differ more than +/- 10% from the average amount/well for the entire filter plate.
Characteristics | GSTrap HP | GSTrap FF | GSTrap 4B |
---|---|---|---|
Chromatography media | Glutathione Sepharose® High Performance | Glutathione Sepharose® 4 Fast Flow | Glutathione Sepharose® 4B |
Average particle size | 34 µm | 90 µm | 90 µm |
Dynamic binding capacity1,2 | Approx. 7 mg rGST/mL medium | Approx. 10 mg rGST/mL medium | Approx. 25 mg horse liver GST/mL medium |
Max. back-pressure3 | 0.3 MPa, 3 bar | 0.3 MPa, 3 bar | 0.3 MPa, 3 bar |
Recommended flow rate3 | Sample loading: 0.2–1 mL/min (1 mL) and 1–5 mL (5 mL) Washing and elution: 1–2 mL/min (1 mL) and 5–10 mL/min (5 mL) | Sample loading: 0.2–1 mL/min (1 mL) and 1–5 mL (5 mL) Washing and elution: 1–2 mL/min (1 mL) and 5–10 mL/min (5 mL) | Sample loading: 0.2–1 mL/min (1 mL) and 0.5–5 mL/min (5 mL) Washing and elution: 1 mL/min (1 mL) and 5 mL/min (5 mL) |
Chemical stability | Stable to all commonly used aqueous buffers, e.g. 1 M acetate, pH 4.0 and 6 M Gua-HCl for 1 h at room temperature | Stable to all commonly used aqueous buffers, e.g. 1 M acetate, pH 6.0, and 6 M Gua-HCl for 1 h at room temperature | Stable to all commonly used aqueous buffers. Exposure to 0.1 M NaOH, 70% ethanol, or 6 M Gua-HCl for 2 h at room temperature or to 1% (w/v) SDS for 14 d causes no significant loss of activity. |
pH stability | 3–12 | 3–12 | 4–13 |
Storage temperature | 4 °C to 30 °C | 4 °C to 30 °C | 4 °C to 8 °C |
Storage buffer | 20% ethanol | 20% ethanol | 20% ethanol |
The column dimensions are identical for all three GSTrap columns (0.7 × 2.5 cm for the 1 mL column and 1.6 × 2.5 cm for the 5 mL column). Column volumes are 1 mL and 5 mL.
1 The binding of GST-tagged proteins depends on size, conformation, and concentration of the protein in the sample loaded. Binding of GST to glutathione is also flow dependent, and lower flow rates often increase the binding capacity. This is important during sample loading. Protein characteristics, pH, and temperature, but also the chromatography medium used may affect the binding capacity.
2 Dynamic binding capacity conditions (60% breakthrough):
Sample: Column volume: Flow rate: Binding buffer: Elution buffer: |
1 mg/mL pure GST-tagged protein in binding buffer 0.4 mL 0.2 mL/min (60 cm/h) 10 mM sodium phosphate, 140 mM NaCl, 2.7 mM KCl, pH 7.4 50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0 |
3 H2O at room temperature.
Chromatography medium | Glutathione Sepharose® 4 Fast Flow |
Column volume | 20 mL |
Column dimensions | 1.6 × 10 cm |
Dynamic binding capacity1,2 | Approx. 200 mg rGST/column |
Recommended flow rate3 | 1–10 mL/min (30–300 cm/h) |
Max. flow rate3 | 10 mL/min (300 cm/h) |
Max. pressure over the packed bed during operation3 | 1.5 bar (0.15 MPa, 22 psi) |
Column hardware pressure limit | 5 bar (0.5 MPa, 73 psi) |
Storage | 20% ethanol |
Storage temperature | 4 °C to 30 °C |
1 The binding of GST-tagged proteins depends on size, conformation, and concentration of the protein in the sample loaded. Binding of GST to glutathione is also flow dependent, and lower flow rates often increase the binding capacity. This is important during sample loading. Protein characteristics, pH, and temperature, but also the chromatography medium used may affect the binding capacity.
2 Dynamic binding capacity conditions (60% breakthrough):
Sample: Column volume: Flow rate: Binding buffer: Elution buffer: |
1 mg/mL pure GST-tagged protein in binding buffer 0.4 mL 0.2 mL/min (60 cm/h) 10 mM sodium phosphate, 140 mM NaCl, 2.7 mM KCl, pH 7.4 50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0 |
3 H2O at room temperature.
Column material frits | Polypropylene barrel, polyethylene |
Column volume | 13 mL |
Medium | Glutathione Sepharose® 4B |
Average bead size | 45–165 µm |
Ligand | Glutathione and 10-carbon linker arm |
Ligand concentration | 7–15 µmol glutathione/mL medium |
Protein binding capacity1 | Approx. 50 mg horse liver GST/column |
Bed volume | 2 mL |
Compatibility during use | All commonly used aqueous buffers |
Chemical stability | No significant loss of the capacity is detected when Glutathione Sepharose® 4B is exposed to 0.1 M citrate (pH 4.0), 0.1 M NaOH, 70% ethanol, or 6 M Gua-HCl2 for 2 h at room temperature. No significant loss of binding capacity is observed after exposure to 1% SDS for 14 d. |
Storage solution | 20% ethanol |
pH stability | 4–13 |
Storage temperature | 4 °C to 30 °C |
Note: It is not recommended to autoclave the columns.
1 Binding capacity is protein dependent. The binding of GST-tagged proteins depends on size, conformation, and concentration of the protein in the sample. Binding of GST to glutathione is also flow dependent, and lower flow rates often increase the binding capacity. This is important during sample loading. Protein characteristics, pH, and temperature may also affect the binding capacity.
2 Exposing the sample to 6 M Gua-HCl will denature the GST-tag. It is therefore important to remove all Gua-HCl before use.
Column material | Polypropylene barrel, polyethylene frits |
Column volume | 900 µL |
Medium | Glutathione Sepharose® 4B |
Average bead size | 90 µm |
Ligand | Glutathione and 10-carbon linker arm |
Ligand concentration | 7–15 µmol glutathione/mL medium |
Protein binding capacity1 | Approx. 500 µg horse liver GST/column |
Bed volume | 50 µL |
Compatibility during use | All commonly used aqueous buffers |
Chemical stability | No significant loss of the capacity is detected when Glutathione Sepharose® 4B is exposed to 0.1 M citrate (pH 4.0), 0.1 M NaOH, 70% ethanol or 6 M Gua-HCl2 for 2 h at room temperature. No significant loss of binding capacity is observed after exposure to 1% SDS for 14 d. |
Storage solution | PBS and 0.05% Kathon CG/ICP Biocide |
pH stability | 4–13 |
Storage temperature | Room temperature |
1 Binding capacity is protein dependent. The binding of GST-tagged proteins depends on size, conformation, and concentration of the protein in the sample loaded. Binding of GST to glutathione is also flow dependent, and lower flow rates often increase the binding capacity. This is important during sample loading. Protein characteristics, pH, and temperature may also affect the binding capacity.
2 Exposing the sample to 6 M Gua-HCl will denature the GST-tag. It is therefore important to remove all Gua-HCl before use.
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