Characteristics of Glutathione Sepharose and HiTrap^® Benzamidine FF (High Sub) Media and Columns

Glutathione Sepharose High Performance is recommended for high-resolution purification of GST-tagged proteins, providing sharp peaks and concentrated eluent. Glutathione Sepharose 4 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 A5.1 summarizes key characteristics of these three Glutathione Sepharose media, and Tables A5.2 to A5.6 summarize the characteristics of these media prepacked in columns and in 96-well filter plates. Table A5.7 summarizes key characteristics of HiTrap Benzamidine Fast Flow (high sub).

Table A5.1 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 GST/ml medium	10 mg recombinant GST/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, e.g., 0.1 M NaOH, 70% ethanol, or 6 M Gua-HCl for 2 h at room temperature or exposure to 1% (w/v) SDS for 14 d.
pH stability	3–12	3–12	4–13
Storage temperature	4 °C to 30 °C	4 °C to 30 °C	4 °C to 30 °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, temperature, and the media used may also affect the binding capacity.

² When using water at room temperature.

A5.2 Characteristics of GST MultiTrap FF and GST MultiTrap 4B

Chromatography media	GST MultiTrap FF: Glutathione Sepharose 4 Fast Flow 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 FF: Up to 0.5 mg GST-tagged protein/well 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: Recommended Maximum	Depends on sample pretreatment and sample properties 100–500 × g 700 × g
Vacuum pressure:  Recommended Maximum	Depends on sample pretreatment and sample properties -0.1 to -0.3 bar -0.5 bar
pH stability	Glutathione Sepharose 4 Fast Flow: 3–12 Glutathione Sepharose 4B: 4–13
Storage	20% ethanol
Storage temperature	4 °C to 30 °C

¹ According to American National Standards Institute (ANSI) and  Society for Biomolecular Screening (SBS).

1-2004, 3-2004, and 4-2004 standards.

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 A5.3 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 column) and 5–10 ml/min (5 ml column)	Sample loading: 0.2–1 ml/min (1 ml) and 1–5 ml (5 ml) Washing and elution:  1–2 ml/min (1 ml column) and 5–10 ml/min (5 ml column)	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 column) and  5 ml/min (5 ml column)
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 30 °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, temperature, and the media used may also affect the binding capacity.

² Dynamic binding capacity conditions (60% breakthrough):

Sample: 1 mg/ml pure GST-tagged protein in binding buffer
Column volume: 0.4 ml
Flow rate: 0.2 ml/min (60 cm/h)
Binding buffer: 10 mM sodium phosphate, 140 mM NaCl, 2.7 mM KCl, pH 7.4
Elution buffer: 50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0

³ When using water at room temperature.

A5.4 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	0% 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, temperature, and the media used may also affect the binding capacity.

² Dynamic binding capacity conditions (60% breakthrough):

Sample: 1 mg/ml pure GST-tagged protein in binding buffer
Column volume: 0.4 ml
Flow rate: 0.2 ml/min (60 cm/h)
Binding buffer: 10 mM sodium phosphate, 140 mM NaCl, 2.7 mM KCl, pH 7.4
Elution buffer: 50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0

³ When using water at room temperature.

Table A5.5 Characteristics of GST GraviTrap prepacked columns

Column material frits	Polypropylene barrel, polyethylene
Column volume	13 ml
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. 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 A5.6 Characteristics of GST SpinTrap columns

Column material frits	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 mg 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	4 °C to 30 °C

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.

A5.7 Characteristics of HiTrap Benzamidine FF (high sub)

Column dimensions (i.d. × h)	0.7 × 2.5 cm (1 ml column) and 1.6 × 2.5 cm (5 ml column)
Column volumes	1 ml and 5 ml
1 ml and 5 ml	p-Aminobenzamidine (pABA)
Spacer	14-atom
Ligand concentration	12 µmol p-Aminobenzamidine/ml medium
Binding capacity	35 mg trypsin/ml medium
Average particle size	90 µm
Bead structure	Highly cross-linked agarose, 4%
Maximum back pressure	0.3 MPa, 3 bar
Recommended flow rates	1 ml/min (1 ml column) and 5 ml/min (5 ml column)
Maximum flow rates	4 ml/min (1 ml column) and 20 ml/min (5 ml column)
Chemical stability	All commonly used aqueous buffers
pH stability short term1	pH 1–9
pH stability long term1	pH 2–8
Storage temperature	4 °C to 8 °C
Storage buffer	20% ethanol in 0.05 M acetate buffer, pH 4

¹ The ranges given are estimates based on our knowledge and experience. Please note the following: pH stability, short term refers to the pH interval for regeneration, cleaning-in-place, and sanitization procedures. pH stability, long term refers to the pH interval where the medium is stable over a long period of time without adverse effects on its chromatographic performance.