Buccutite™ Streptavidin Antibody Conjugation Kit *Optimized for Labeling 25 ug Protein*

1. Add 1.25 µL Reaction Buffer (Component C) into antibody (25 µL).

2. Add 2.5 µL reconstituted Buccutite™ MTA (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 4°C. Proper storage is important to ensure that the kit remains stable for up to six months. Alternatively, Components A and B can be stored at -20°C. However, please do not freeze the Reaction Buffer (Component C). Before opening the vials, it is recommended to warm all the components and briefly centrifuge them. Then, immediately prepare the required solutions before starting the conjugation process. The following SOP provides an example of how to label goat anti-mouse IgG antibodies.

1. To label 25 µg of antibody (assuming the target antibody concentration is 1 mg/mL), mix 1.25 µL (5% of the total reaction volume) of Reaction Buffer (Component C) with 25 µL of the target antibody solution.

   Note: If you have a different concentration, adjust the antibody volume accordingly to make ~25 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.

1. Add 10 µL DMSO (not provided in the kit) into the vial of Buccutite ™ MTA.

1. Add 2.5 µL of the Buccutite™ MTA working solution to the antibody working solution. Mix thoroughly by pipetting or vortexing.

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 to the vial of the Buccutite™ FOL-Acitvated Streptavidin (Component A). The total volume should be 27.5 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 at room temperature.

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

   Note: The antibody concentration is 0.91 mg/mL.

The antibody-streptavidin conjugate should be stored at > 0.5 mg/mL in the presence of a carrier protein (e.g., 0.1% bovine serum albumin). For longer storage, the antibody-streptavidin conjugates could be lyophilized and stored at ≤ –20 °C.