1) Dissolve HRP in phosphate buffered saline, pH 7.0 (without adding preservative like sodium azide) at 10mg/mL

2) Add freshly prepared glutaraldehyde to a final concentration of 1.5% (w/v). Mix overnight at room temperature.

3) Purify the activated enzyme by gel filtration using Sephadex G25 column and the same buffer.

4) Dissolve streptavidin in 0.5M sodium carbonate buffer, pH 9.5 at 10mg/mL. Add the activated HRP to the streptavidin solution using a molar ratio of 2moles of HRP per 1 mole of streptavidin.

Mix overnight at 4oC.

5) Block by adding 50uL of 0.2M lysine for each mL of the conjugate solution. Incubate for 2hours at room temperature

6) Centrifuge to remove any precipitate then purify by dialysis or gel filtration.

Thank you very much Ahmed 🙏🙏🌹