Buccutite™ Rapid trFluor™ D2 Acceptor Antibody Labeling Kit *Microscale Optimized for Labeling 100 ug Antibody Per Reaction*

1. Add 5 µL Reaction Buffer (Component C) into the antibody solution (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 trFluor™ D2 working solution.

5. Incubate at room temperature for 60 minutes.

Please store the kit at 4°C upon receiving it. Ensure it is stored properly to maintain stability for six months. Alternatively, Component B can be stored at -20°C. Do not freeze Buccutite™ FOL-Activated trFluor™ D2 (Component A) or Reaction Buffer (Component C). Before opening the vials, warm all components and briefly centrifuge them. Prepare the required solutions immediately after opening the vials to begin your conjugation. For reference, an example SOP for labeling goat anti-mouse IgG antibodies is provided.

For labeling 100 µg antibody (assuming the target antibody concentration is 1 mg/mL), mix 5 µL (5% of the total reaction volume) of 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 Buccutite™ FOL-Activated trFluor™ D2 solution by adding 50 µL ddH2O into the vial of Buccutite™ FOL-Activated trFluor™ D2 (Component A), mix well by repeatedly pipetting for a few times or vortex the vial for a few seconds.

2. Mix the whole vial of Buccutite™ FOL-Activated trFluor™ D2 solution into the antibody-Buccutite™ MTA solution, mix well, and rotate the mixture for 1 hour at room temperature.

3. The antibody-trFluor™ D2 conjugate is now ready to use.

   Note: For immediate use, the antibody-trFluor™ D2 conjugate needs to 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-trFluor™ D2 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-trFluor™ D2 conjugates could be lyophilized and stored at ≤ –20 °C.