Streptavidin–HRP (Detection B ) Preparation Process
1. Dissolve HRP in water , at a concentration of 8 mg/ml
2. Dissolve sodium periodate in water at a concentration of 0.18 M
3. Add 200 ul sodium periodate solution to 1 ml HRP solution, Mix to dissolve, Protect from light.
4. In the dark, react for 25 minutes at room temperature. A color change will be apparent as the reaction proceeds—changing from the brownish/gold color of concentrated HRP to green.
5. Purify the oxidized enzyme , we could dialyze and desalt in PBS.
6. Dissolve streptavidin at a concentration of 8 mg/ml in 0.1 M CBS, PH 9.6, at room temperature.
7. The periodate-oxidized HRP (prepared above) is finally purified by 0.1 M CBS, PH 9.6
8. Mix the (strept)avidin solution with the enzyme solution at a ratio of 1:4(v/v).
9. React for 2-4 hours at room temperature to form the initial Schiff base interactions.
10. In a bottle, add 20 ul of 2 M sodium cyanoborohydride of 1ml reaction solution
11. React for 30-60 minutes at room temperature
12. Block unreacted aldehyde sites by addition 50ul 1 M ethanolamine
13. React for 30-60 minutes at room temperature.
14. Purify the compound by Sephadex G-75, then dialyze and desalt in PBS.
2. Dissolve sodium periodate in water at a concentration of 0.18 M
3. Add 200 ul sodium periodate solution to 1 ml HRP solution, Mix to dissolve, Protect from light.
4. In the dark, react for 25 minutes at room temperature. A color change will be apparent as the reaction proceeds—changing from the brownish/gold color of concentrated HRP to green.
5. Purify the oxidized enzyme , we could dialyze and desalt in PBS.
6. Dissolve streptavidin at a concentration of 8 mg/ml in 0.1 M CBS, PH 9.6, at room temperature.
7. The periodate-oxidized HRP (prepared above) is finally purified by 0.1 M CBS, PH 9.6
8. Mix the (strept)avidin solution with the enzyme solution at a ratio of 1:4(v/v).
9. React for 2-4 hours at room temperature to form the initial Schiff base interactions.
10. In a bottle, add 20 ul of 2 M sodium cyanoborohydride of 1ml reaction solution
11. React for 30-60 minutes at room temperature
12. Block unreacted aldehyde sites by addition 50ul 1 M ethanolamine
13. React for 30-60 minutes at room temperature.
14. Purify the compound by Sephadex G-75, then dialyze and desalt in PBS.
