Procedure for extracting bacterial proteins using the T7• Tag Affinity Purification Kit
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.
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Ceftriaxone sodium powder is called preparation, sterile ceftriaxone sodium is called API, and 7-ACA is called intermediate.
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