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 A2.1Characteristics 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.

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.

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.

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.

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.

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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