Buccutite™ Streptavidin 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 Streptavidin (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 10 µL of DMSO (not provided) into the vial of Buccutite ™ MTA (Component B).

1. Add the antibody working solution directly into the vial of Buccutite™ MTA (Component B), and mix them well by repeatedly pipetting a few times or vortexing 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. Add the antibody-Buccutite™ MTA reaction mixture directly into the vial of Buccutite™ FOL-Activated Streptavidin (Component A). The total volume should be 200 µL. After adding, mix well by repeatedly pipetting a few times or vortexing the vial for a few seconds.

2. Incubate for 1-2 hours.

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

   Note: The antibody concentration is 0.5 mg/mL.

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 Streptavidin-Antibody conjugate solution could be stored at 4 °C for two months without significant change and kept from light. For longer storage, the Streptavidin-antibody conjugates could be lyophilized and stored at ≤ –20 °C.