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| Description:Glutathione S-transferase (GST) gene fusion systems have been widely used for obtaining large amounts of desirable protein in Escherichia coli. The fusion protein, which contains a GST tail can then be purified through affinity chromatography. MCLABs glutathione agarose is designed for the specific purification of GST recombinant proteins and other glutathione-binding proteins. Glutathione agarose is uniquely formulated for excellent binding capacity and purity of the protein of interest.Protocol:The following instructions for GST-fusion protein purification can be scaled up or down depending on the users preference. This manual exemplifies sample preparation from a specific amount of starting material and purification using 1 ml resin.1. Centrifuge sample after cell lysis to remove undissolved membranes and cellular debris before applying to purification column. 2. Wash purification column with 10x bead volume of Binding Buffer to remove azide.3. Dilute an appropriate amount sample with a 1:1 ratio of Binding Buffer before applying to purification column.4. Wash the purification column with 10x bead volume of Binding Buffer or until no proteins can be detected in the washes.5. Elute bound protein of interest with 5x bead volume of Elution Buffer.6. GST agarose beads can be saved for later use by washing the purification column with Binding Buffer containing 3 M NaCl. After a thorough wash, the purification column should be equilibrated in Binding Buffer containing 2 mM sodium azide and stored at 4°C. GST-fusion proteins are generally eluted from glutathione agarose beads with the use of excess glutathione. Alternatively, GST-fusion protein can be encoded with a cleavage site between the GST and the protein, allowing the desirable protein to be eluted with the use of a protease. Recommended Storage Conditions: -20ºC.Binding Buffer: 50 mM Tris150 mM NaClpH 8.0Elution Buffer:50 mM Tris pH 7.80.15 M NaCl1 mM EDTA1 mM DTT10 mM GST |
