These three chromatography media are all used for the puriﬁcation of GST-tagged recombinant proteins and other S-transferases or glutathione-dependent proteins. They allow mild elution conditions that preserve protein structure and function. All are supplied preswollen in 20% ethanol and are also available in various prepacked formats, such as GSTrap, as described later in this chapter. Appendix 2 (Characteristics of Glutathione Sepharose products) for the main characteristics of all Glutathione Sepharose media.
In Glutathione Sepharose High Performance, the glutathione ligand is coupled to highly cross-linked 6% agarose. The medium has an average bead size of 34 µM and can be used for high-resolution puriﬁcation and elution of a more concentrated sample.
In Glutathione Sepharose 4 Fast Flow, the glutathione ligand is coupled to highly cross-linked 4% agarose. The medium has an average bead size of 90 µM. It is a good choice for scale-up due to its good binding capacity and ﬂow properties. This medium is also suitable for batch and gravity-ﬂow puriﬁcations.
In Glutathione Sepharose 4B, the glutathione ligand is coupled to 4% agarose. The medium has an average bead size of 90 µM. It provides very high binding capacity and is recommended for small-scale puriﬁcation as well as batch and gravity-ﬂow operations.
Glutathione Sepharose 4 Fast Flow and Glutathione Sepharose 4B are also available prepacked in 96-well ﬁlter plates.
Procedures for both batch and column puriﬁcation of GST-tagged proteins follow.
Figure 5.3.Glutathione Sepharose High Performance, Glutathione Sepharose 4 Fast Flow, and Glutathione Sepharose 4B for puriﬁcation of GST-tagged proteins.
Refer to General considerations for puriﬁcation of GST-tagged proteins for general considerations before beginning this procedure.
Adjust the sample to the composition and pH of the binding buffer by additions from concentrated stock solutions; by diluting the sample with binding buffer; or by buffer exchange.
Pass the sample through a 0.22 µM or a 0.45 µM ﬁlter and/or centrifuge it immediately before sample application. If the sample is too viscous, dilute it with binding buffer to prevent it from clogging; increase lysis treatment (sonication, homogenization); or add DNase/RNase to reduce the size of nucleic acid fragments.
Use high-purity water and chemicals, and ﬁlter all buffers through a 0.45 µM ﬁlter before use.
1 to 20 mM DTT may be included in the binding and elution buffers to reduce the risk of oxidation of free -SH groups on GST, which may cause aggregation of the tagged target protein, resulting in lower yield of GST-tagged protein.
Glutathione Sepharose chromatography media are supplied preswollen in 20% ethanol. The media are used at a ﬁnal slurry concentration of 50%.
Glutathione Sepharose media must be thoroughly washed with PBS to remove the ethanol storage solution because residual ethanol may interfere with subsequent procedures.
For cleaning, storage, and handling information, refer to Appendix 2 (Characteristics of Glutathione Sepharose products).
See instructions supplied with the products or refer to Appendix 6 (Column packing and preparation) for general guidelines for column packing.
For recommended ﬂow rates, Appendix 6 (Column packing and preparation), Table A6.6.
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