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).

Table A2.1 Characteristics of 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.

Table A2.2 Characteristics of GST MultiTrap 4B

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.

Table A2.3 Characteristics of prepacked GSTrap HP, GSTrap FF, and GSTrap 4B columns

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.

Table A2.4 Characteristics of GSTPrep FF 16/10

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.

Table A2.5 Characteristics of GST GraviTrap prepacked columns

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.

Table A2.6 Characteristics of GST SpinTrap columns

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.