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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).

CharacteristicsGlutathione Sepharose®  High PerformanceGlutathione Sepharose® 4 Fast FlowGlutathione Sepharose® 4B
MatrixHighly cross-linked 6% agaroseHighly cross-linked 4% agarose4% agarose
Average particle size34 µm90 µm90 µm
Ligand concentration1.5–3.5 mg glutathione/mL medium (based on Gly)120–320 µmol glutathione/mL medium200–400 µmol glutathione/g washed and dried medium
Binding capacity17 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/h50–300 cm/h< 75 cm/h
Chemical stabilityStable to all commonly used aqueous buffers, e.g. 1 M acetate, pH 4.0 and 6 M Gua-HCl for 1 h at room temperatureStable to all commonly used aqueous buffers, e.g. 1 M acetate, pH 4.0, and 6 M Gua-HCl for 1 h at room temperatureStable 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 stability3–123–124–13
Storage temperature4 °C to 30 °C4 °C to 30 °C4 °C to 8 °C
Storage buffer20% ethanol20% ethanol20% ethanol
Table A2.1Characteristics of Glutathione Sepharose® High Performance, Glutathione Sepharose® 4 Fast Flow, and Glutathione Sepharose® 4B

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 mediaGST MultiTrap 4B: Glutathione Sepharose® 4B
Filter plate size1127.8 × 85.5 × 30.6 mm
Filter plate materialPolypropylene and polyethylene
Binding capacityGST MultiTrap 4B: Up to 0.5 mg GST-tagged protein/well
Reproducibility between wells2+/- 10%
Volume packed medium/well50 µL (500 µL of 10% slurry)
Number of wells96
Centrifugation speed:Depends on sample pretreatment and sample properties
           recommended100–500 × g
           maximum700 × g
Vacuum pressure:Depends on sample pretreatment and sample properties
           recommended-0.1 to -0.3 bar
           maximum-0.5 bar
pH stabilityGlutathione Sepharose® 4B: 4–13
Storage20% ethanol
Storage temperature4 °C to 8 °C
Table A2.2Characteristics of GST MultiTrap 4B

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.

CharacteristicsGSTrap HPGSTrap FFGSTrap 4B
Chromatography mediaGlutathione Sepharose® High PerformanceGlutathione Sepharose® 4 Fast FlowGlutathione Sepharose® 4B
Average particle size34 µm90 µm90 µm
Dynamic binding capacity1,2Approx. 7 mg rGST/mL mediumApprox. 10 mg rGST/mL mediumApprox. 25 mg horse liver GST/mL medium
Max. back-pressure30.3 MPa, 3 bar0.3 MPa, 3 bar0.3 MPa, 3 bar
Recommended flow rate3Sample 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 stabilityStable to all commonly used aqueous buffers, e.g. 1 M acetate, pH 4.0 and 6 M Gua-HCl for 1 h at room temperatureStable to all commonly used aqueous buffers, e.g. 1 M acetate, pH 6.0, and 6 M Gua-HCl for 1 h at room temperatureStable 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 stability3–123–124–13
Storage temperature4 °C to 30 °C4 °C to 30 °C4 °C to 8 °C
Storage buffer20% ethanol20% ethanol20% ethanol
Table A2.3Characteristics of prepacked GSTrap HP, GSTrap FF, and GSTrap 4B columns

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 mediumGlutathione Sepharose® 4 Fast Flow
Column volume20 mL
Column dimensions1.6 × 10 cm
Dynamic binding capacity1,2Approx. 200 mg rGST/column
Recommended flow rate31–10 mL/min (30–300 cm/h)
Max. flow rate310 mL/min (300 cm/h)
Max. pressure over the packed  bed during operation31.5 bar (0.15 MPa, 22 psi)
Column hardware pressure limit5 bar (0.5 MPa, 73 psi)
Storage20% ethanol
Storage temperature4 °C to 30 °C
Table A2.4Characteristics of GSTPrep FF 16/10

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 fritsPolypropylene barrel, polyethylene
Column volume13 mL
MediumGlutathione Sepharose® 4B
Average bead size45–165 µm
LigandGlutathione and 10-carbon linker arm
Ligand concentration7–15 µmol glutathione/mL medium
Protein binding capacity1Approx. 50 mg horse liver GST/column
Bed volume2 mL
Compatibility during useAll commonly used aqueous buffers
Chemical stabilityNo 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 solution20% ethanol
pH stability4–13
Storage temperature4 °C to 30 °C
Table A2.5Characteristics of GST GraviTrap prepacked columns

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 materialPolypropylene barrel, polyethylene frits
Column volume900 µL
MediumGlutathione Sepharose® 4B
Average bead size90 µm
LigandGlutathione and 10-carbon linker arm
Ligand concentration7–15 µmol glutathione/mL medium
Protein binding capacity1Approx. 500 µg horse liver GST/column
Bed volume50 µL
Compatibility during useAll commonly used aqueous buffers
Chemical stabilityNo 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 solutionPBS and 0.05% Kathon CG/ICP Biocide
pH stability4–13
Storage temperatureRoom temperature
Table A2.6Characteristics of GST SpinTrap 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 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.

Materials
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