Buccutite™ Peroxidase (HRP) Antibody Conjugation Kit *Optimized for Labeling 100 ug Protein*

1. Add 5 µL Reaction Buffer (Component C) into antibody (100 µL)

2. Add the antibody solution into Buccutite™ MTA vial (Component B)

3. Incubate at room temperature for 30 minutes

4. Mix with 50 µL Buccutite™ FOL-Activated HRP (Component A)

5. Incubate at room temperature for 60 minutes

Upon receiving the kit, it should be stored at a temperature of 4°C. When stored properly, the kit will remain stable for a period of six months. Alternatively, it is possible to store components A and B at a temperature of -20°C. Before opening the vials, it is recommended to warm all the components and briefly centrifuge them. Afterward, proceed to immediately prepare the required solutions before starting your conjugation. The following SOP serves as an example for labeling goat anti-mouse IgG antibody.

1. To label 100 µg of antibody (assuming the target antibody concentration is 1 mg/mL), mix 5 µL (5% of the total reaction volume) of the Reaction Buffer (Component C) with 100 µL of the target antibody solution.

   Note: If you have a different concentration, adjust the antibody volume accordingly to make ~100 µg antibody available for your labeling reaction.

   Note: The antibody should be dissolved in 1X phosphate-buffered saline (PBS), pH 7.2-7.4. If the antibody is dissolved in glycine buffer, it must be dialyzed against 1X PBS, pH 7.2-7.4, or use ReadiUse™ 10KD Spin Filter (Cat. #60502 from AAT Bioquest) to remove free amines or ammonium salts (such as ammonium sulfate and ammonium acetate) that are widely used for antibody precipitation.

   Note: Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or gelatin will not be labeled well.

   Note: The antibody –Buccutite™ MTA reaction efficiency is significantly reduced if the antibody concentration is less than 1 mg/mL. For optimal labeling efficiency, the final antibody concentration range of 1-10 mg/mL is recommended.

1. Add the antibody working solution directly into the vial of Buccutite ™ MTA (Component B), and mix them well by repeatedly pipetting for a few times or vortex the vial for a few seconds.

2. Keep the antibody- Buccutite ™ MTA reaction mixture at room temperature for 30 - 60 minutes.

   Note: The antibody-Buccutite™ MTA reaction mixture can be rotated or shaken for a longer time if desired.

1. Make HRP- Buccutite™ FOL solution by adding 50 µL ddH₂O into the vial of Buccutite™ FOL-Activated HRP (Component A), mix well by repeatedly pipetting for a few times or vortex the vial for a few seconds.

2. Mix whole vial of Buccutite™ FOL-Activated HRP solution into the antibody- Buccutite™ MTA solution, mix well and rotating the mixture for 1 hour at room temperature.

3. The HRP-antibody conjugate is now ready to use.

   Note: For immediate use, dilute the HRP-antibody conjugate with a buffer of your choice.

   Note: Alternatively, add antibody-Buccutite™ MTA solution mixture to the vial of Buccutite™ FOL-Activated HRP directly.

The antibody conjugate should be stored at > 0.5 mg/mL in the presence of a carrier protein (e.g., 0.1% bovine serum albumin). The HRP-Antibody conjugate solution could be stored at 4 °C for two months without significant change and kept from light. For longer storage, the HRP-antibody conjugates could be lyophilized and stored at ≤ –20 °C.