Extraction and purification technology of proteins in bacteria

Experimental reagent

T7• Tag Affinity Purification Kit

T7•Tag antibody agar. B/W buffer: 4.29 mM Na2HPO4, 1.47 mM KH2PO4, 2.7 mM KCl, 0.137 mM NaCl, 1% Tween-20, pH 7.3 Elution buffer: 0.1 M citric acid, pH 2.2. Neutralization buffer : 2M Tris, pH 10.4. PEG 20000.

Experimental procedure

1.100 ml of the cells containing the recombinant expression plasmid were induced, centrifuged at 5000 g for 5 min, the supernatant was discarded, and the cells were harvested and resuspended in 10 ml of pre-cooled B/W buffer.

2. The suspension was sonicated on ice until the sample was no longer viscous. Centrifuge at 14000 g for 30 min at 4 ° C. The supernatant was taken and filtered through a 0.45 μm membrane as a sample solution.

3. The agar bound to the T7•Tag antibody was fully suspended, equilibrated to room temperature, and loaded into a column.

4. After the B/W buffer is equilibrated, the sample solution passes through the column.

5. 10 ml of B/W buffer was passed through the column and the unbound protein was washed away.

6. Using 5 ml of elution buffer, 1 ml each time, the eluate was collected in a centrifuge tube containing 150 μl of neutralization buffer, mixed and placed on ice, and analyzed by SDS-PAGE.

7. Place the eluted protein in a dialysis bag, dialyze on double distilled water for 24 hr, and change the solution several times in the middle.

8. Concentrate the protein with PEG 20000.

Precautions

The protein is filtered through a 0.45 μm membrane before passing through the column, otherwise there will be insolubles in the column after several purifications.

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