GST protein purification step

Prepare cell lysates:

1. The cell pellet of each 100 ml culture was suspended in 4 ml of PBS;

2. Add lysozyme to a final concentration of 1 mg/ml, and place on ice for 30 min;

3. Forcibly inject 10 ml of 0.2% Triton X- 100 into the cell lysate with a syringe and mix vigorously several times;

4. Add DNase and RNase to a final concentration of 5 μg/ml, and incubate for 10 min at 4 °C;

5.4 ° C 3000g (5000r / min) centrifugation for 30min;

6. Transfer the supernatant to a new tube and add DTT to a final concentration of 1 mmol/L.

Purified fusion protein:

7. Cell lysate is mixed with 50% glutathione-agarose resin homogenate, add 2ml resin per 100ml cell culture, and shake gently for 0min at room temperature;

8. The mixture was centrifuged at 500 g (2100 r/min) for 5 min at 4 ° C, the supernatant was carefully removed and a small amount of SDS-PAGE was taken;

9. Add 10 times the standard volume of PBS to the precipitate, invert the centrifuge tube several times to mix, wash away the protein not bound to the resin;

Centrifuge at 500g (2100r/min) for 5min at 10.4°C, carefully remove the supernatant and leave a little for SDS-PAGE;

11. Repeat steps 9 and 10 twice;

12. The bound GST fusion protein can be eluted with glutathione elution buffer and can also be cleaved with thrombin , enterokinase or factor Xa to release the target protein.

Eluting the fusion protein with glutathione:

a. Add 1 column bed volume of glutathione elution buffer to the precipitate, gently agitate for 10 min at room temperature, and elute the bound protein on the resin;

b. Centrifuge at 500g (2100r/min) for 5min at 4°C, and move the supernatant to a new tube;

c. Repeat steps a and b twice and combine the supernatants for 3 times.

Proteolysis recovers target cells from the bound GST fusion protein:

a. Add thrombin, enterokinase or factor Xa to the resin that incorporates the fusion protein. Add 50 units of protease dissolved in 1 ml of PBS per ml of resin, invert the tube several times, mix at room temperature for 2-16 h, and determine the precise time by small-scale experiment;

b. Centrifuge at 500g (2100r/min) for 5min at 4°C, and move the supernatant to a new tube;

13.10% SDS-PAGE analysis of the protein composition of each step (cell extraction, washing and elution) of the sample.

Treatment of glutathione agarose resin:

1. Gently invert the container containing glutathione-agarose resin and mix the resin into a homogenate;

2. Take a portion of the homogenate into a 15 ml polypropylene tube (2 ml homogenate per 100 ml of bacterial culture);

Centrifuge at 500g (2100r/min) for 5min at 3.4°C, carefully remove the supernatant;

4. Add 10 times of bed volume of cold PBS to the resin, invert several times, mix the homogenate, centrifuge at 500g (2100r/min) for 5min at 4°C, carefully remove the supernatant;

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