Buccutite™ Rapid PE-iFluor® 594 Tandem Antibody Labeling Kit *Microscale Optimized for Labeling 100 ug Antibody Per Reaction*

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 PE-iFluor® 594 (Component A)

5. Incubate at room temperature for 60 minutes

Important: Store the kit at 4 °C upon receipt. When stored correctly, the kit remains stable for six months. Alternatively, Component B can be stored at -20 °C. Avoid freezing Buccutite™ FOL-Activated PE-iFluor® 594 (Component A) and Reaction Buffer (Component C). Before use, warm all components to room temperature and briefly centrifuge the vials before opening. Prepare the necessary solutions immediately prior to starting the conjugation. The following SOP provides an example for labeling goat anti-mouse IgG antibody.

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

   Note: If your antibody has a different concentration, adjust the volume to ensure approximately 100 µg of antibody is available for the 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 longer time if desired.

1. Make Buccutite™ FOL-Activated PE-iFluor® 594 solution by adding 50 µL ddH2O into the vial of Buccutite™ FOL-Activated PE-iFluor® 594 (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 PE-iFluor® 594 solution into the antibody-Buccutite™ MTA solution, mix well and rotating the mixture for 1 hour at room temperature.

3. The antibody-PE-iFluor® 594 conjugate is now ready to use.

   Note: For immediate use, the antibody-PE-iFluor® 594 conjugate need be diluted with the buffer of your choice.

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 Antibody-PE-iFluor® 594 conjugate solution could be stored at 4 °C for two months without significant change when stored in the presence of 2 mM sodium azide and kept from light. For longer storage, the antibody-PE-iFluor® 594 conjugates could be lyophilized and stored at ≤ –20 °C.

Table 1. Available fluorophores at AAT Bioquest Buccutite™ Rapid Antibody Labelling Kits