Buccutite™ Rapid PE-iFluor® 594 Tandem Antibody Labeling Kit *Microscale Optimized for Labeling 25 ug Antibody Per Reaction*

1. Add 1.25 µL Reaction Buffer (Component C) into antibody (25 µL)
2. Add 2.5 µL Buccutite™ MTA working solution
3. Incubate at room temperature for 30 - 60 minutes

4. Mix with 50 µL Buccutite™ FOL-Activated PE-iFluor® 594 working solution

5. Incubate at room temperature for 60 minutes

Important: Upon receipt, store the kit at 4 °C. When stored properly, the kit should be stable for six months. Alternatively Components A and B can be stored at -20 °C. Do not freeze Reaction Buffer (Component C). Warm all the components and centrifuge the vials briefly before opening, and immediately prepare the required solutions before starting your conjugation. The following SOP is an example for labeling goat anti-mouse IgG antibody.

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 your antibody has a different concentration, adjust the volume to ensure approximately 25 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 Amicon Ultra-0.5, Ultracel-10 Membrane, 10 kDa (Cat. #UFC501008 from Millipore) to remove free amines or ammonium salts (such as ammonium sulfate and ammonium acetate) that are widely used for antibody precipitation.

   Note: Ensure the antibody is pure and not stabilized with bovine serum albumin (BSA) or gelatin, as these stabilizers significantly impair labeling efficiency.

   Note: For optimal labeling efficiency, the antibody concentration should be in the range of 1–10 mg/mL. If the concentration is below 1 mg/mL, the Buccutite™ MTA reaction efficiency is significantly reduced.

1. Add 10 µL of DMSO (Not provided) to the vial of Buccutite™ MTA (Component B).

1. Add 50 µL of ddH₂O to the vial of Buccutite™ FOL-Activated PE-iFluor® 594 (Component A).

1. Add 2.5 µL of Buccutite ™ MTA working solution into antibody working solution, 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. Add 50 µL of Buccutite™ FOL-Activated PE-iFluor® 594 working solution with Antibody-Buccutite™ MTA solution, mix well by repeatedly pipetting for a few times or vortex the vial for a few seconds.

2. Incubate for 1 to 2 hours.

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

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

   Note: For longer term storage, antibody-PE-iFluor® 594 conjugate solution must be concentrated or freeze dried.

The antibody conjugate should be stored in the presence of a carrier protein (e.g., 0.1% bovine serum albumin) and 0.02-0.05% sodium azide. The Ab-PE-iFluor® 594 conjugate solution could be stored at 4 °C for two months without significant change and kept from light.

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