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Product Description:
Foreign proteins expressed by pGEX vectors are fused with glutathione S-transferase and can therefore be purified by glutathione-agarose affinity chromatography. GSTs are a class of enzymes that use glutathione (gamma-glutamylcysteinylglycine) as a substrate to inactivate toxic small molecules by forming mercaptan uric acid. Since the affinity of GST to the substrate is submillimolar, the affinity chromatography resin formed by glutathione solidified in agarose has a high purification efficiency for GST and its fusion protein. A buffer containing free glutathione can be used to elute the binding GST fusion protein. The resin was regenerated with a buffer of 3mol/L NaCl. Glutathione agarose has a strong binding capacity for GST fusion protein (8 mg of fusion protein per ml of column bed volume).
Characteristics : Particle size: 45-165μm Flow rate: 75cm/h Working PH: 4-10 Pressure: 0.3Mpa Load: > 10mg glutathione S-transferase
Instructions for use:
Treatment of glutathione resin :
1. Gently invert the container containing glutathione-agarose resin and blend the resin into a homogenate.
2. Take part of the homogenate and put it into 15mL polypropylene tube (about 2mL homogenate is required for every 100mL bacterial culture).
Centrifuge at 3.4℃at 500 g for 5 minutes, carefully remove the supernatant.
4. Add PBS pre-cooled by 10 times the volume of the column bed into the resin, mix it upside down several times, centrifuge at 4℃500 g for 5 minutes, and carefully remove the supernatant.
5. Add 1 ml of cold PBS per ml of resin to make 50% homogenate, reverse several times, mix evenly, and put the suspension on ice for use. Preparation of cell extracts:
6. Cell precipitates per 100 ml of culture were resuspended in 4mL of PBS buffer.
7. Add lysozyme until the final concentration is 1mg/mL and leave on ice for 30 minutes.
8. Forced injection of 10mL of 0.2% TritonX-100 into the viscous cell lysate with a syringe and mixed with vigorous vibration for several times. DNase and RNase were added to the final concentration of 5ug/mL, incubated by vibration at 4℃for 10 minutes, centrifuged at 3000 g at 4℃for 30 minutes, the insoluble cell debris was removed, the supernatant was transferred to a new tube, and DTT was added to the final concentration of 1mmol/L. Purification of fusion protein:
9. The cell lysates were mixed with an appropriate amount of 50% glutathione-agarose resin homogenate, 2mL resin per 100ml of bacterial culture, and gently shaken at room temperature for 30min.
10. The mixture was centrifuged at 500 g at 4℃for 5 min. The supernatant was carefully removed and a little sample was left for SDS polyacrylamide gel electrophoresis.
Elution of fusion protein with glutathione:
1. Add cold PBS, 10 times the volume of the column bed, and mix it in the reverse centrifuge tube several times to wash away the protein that is not bound with the resin.
Centrifuge at 500 g at 2.4℃for 5 min, remove the supernatant carefully and leave a little sample for SDS polyacrylamide gel electrophoresis.
3. Repeat Steps 11 and 12 twice.
4. The bound GST fusion protein can be eluted with glutathione, or cut with thrombin, enterkinase or factor Xa to release the target protein.
5. Glutathione eluting buffer of 1 times the volume of the column bed was added to the precipitation, and gently stirred at room temperature for 10min to eluate the bound proteins on the resin.
Centrifuge at 500 g at 6.4℃for 5 min, the supernatant (containing eluted fusion protein) was transferred to the new tube.
7. Repeat steps a and b twice to merge the supernatant three times. Proteolytic hydrolysis recovers the target protein from the bound GST fusion protein:
8. Add thrombin, intestinal kinase or factor Xa (depending on site selection in the fusion protein) to the resin bound with the fusion protein, and add 50 units of protease dissolved in 1mLPBS per mL of resin. Mix the inverted centrifugal tube several times and shake at room temperature for 2-16 hours. Small scale experiments to determine the exact time.
Centrifuge at 500 g at 9.4℃for 5 min. Carefully transfer the supernatant to the new tube. (GST is still bound to the resin, while the target protein is in the supernatant, and the protease is also in the supernatant, which still needs to be separated)
10.10%SDS polyacrylamide gel electrophoresis was used to analyze the protein composition of each step. Reagent preparation: Glutathione eluting buffer: 10mmol/L reduced glutathione, 50mmol/L Tris-Cl (pH8.0)
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