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

Extracted from GST Gene Fusion System, GE Healthcare, 2014

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.

Table 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: 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 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.

Table 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 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, 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 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 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.

Table 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
Ligand 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.

Materials

     
Related Links

Related Protocols