lution of GST-Fusion Protein from Glutathione Agarose

lution of GST-Fusion Protein from Glutathione Agarose

1. Wash the GST-fusion protein glutathione-agarose complex once with 1% Triton X-100 in PBS.

2. Resuspend the complex in 1% Triton X-100 in PBS.

3. Prepare a small column (e.g. a glass or plastic serological pipette with tip plugged with siliconized glass wool) and pour slurry into column.

4. After the beads have settled, flush the column with two column volumes of 1% Triton X-100 in PBS.

5. Wash the column with two volumes of 150 mM NaCl in 50 mM Tris-HCl, pH8.

6. Elute the GST fusion protein from the column material with 1 volume of 20 mM glutathione and 150 mM NaCl in 50 mM Tris-HCl, pH 8.

The total volume of the flow-through will be 3 to 5 ml and should be collected in 0.5 ml aliquots. Approximately 90% of the fusion protein will be collected in 1 to 2 of these aliquots and can be monitored by gel electrophoresis and silver staining.

To remove the glutathione and to concentrate the eluted protein, the solution can either be subjected to ultrafiltration (e.g. using a Centricon 10TM unit), or the protein can be precipitated using trichloroacetic acid (denaturing) or ammonium sulfate (final concentration of 80%, non-denaturing).