ReadiLink™ Rapid iFluor® 647 Antibody Labeling Kit *Production Scale*

1. 1.0 mg Antibody (MW ~150 kDa)

2. Antibody concentration: 2.0 mg/mL

3. Antibody volume: 500 µL

Before opening the vials, warm all components and briefly centrifuge. Immediately prepare necessary solutions before starting conjugation. This protocol is a recommendation.

1. Warm up a vial of reactive dye (Component A) to room temperature.

   Note: Each vial of reactive dye contains an optimized amount of dye to label 1 mg of IgG (MW ~150 kDa) at 2 mg/mL in PBS, the kit can also be used to label other proteins (>10 kDa).

2. Add 10 µL of DMSO (Component D) to the vial of reactive dye (Component A), mix well.

3. Prepare a 500 µL antibody solution in PBS with a concentration of 2 mg/mL.

   Note: The protein should be dissolved in 1X phosphate buffered saline (PBS), pH 7.2 - 7.4. If the protein is dissolved in buffers containing primary amines, like Tris and/or glycine, it must be dialyzed against 1X PBS, pH 7.2 - 7.4, or use Amicon Ultra0.5, Ultracel-10 Membrane, 10 kDa (Cat No. UFC501008 from Millipore) to remove free amines or ammonium salts (such as ammonium sulfate and ammonium acetate) that are widely used for protein precipitation.

   Note: Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or gelatin will not be labeled well.

4. Add 25 µL of Reaction Buffer (Component B) to the antibody solution.

5. Transfer the reconstituted dye solution into the vial of antibody solution, and pipette several times to mix well.

6. Rotate the reaction mixture for 1 hour at room temperature.

1. Twist off the bottom closure of the desalting column (Component D), and loosen the cap. Place the column in a collection tube.

2. Centrifuge the column at 1,000 g for 2 minutes to remove the storage solution.

3. Remove the cap and slowly add 1 mL of PBS to the column. Centrifuge at 1,000 g for 2 minutes and remove the buffer. Repeat this step 3 additional times, discarding the buffer from the collection tube each time.

4. Place the column in a new collection tube, and gently apply the sample into the center of the compact resin bed.

5. Centrifuge the column at 1,000 g for 2 minutes to collect the sample.

The following formula can be used to calculate the antibody concentration:

(A₂₈₀ - CF₂₈₀ x A_dye) / 1.4

The following formula can be used to calculate the degree of labeling:

DOL = (A_dye / Ec_dye) / (A₂₈₀ - CF₂₈₀ x A_dye) / 210,000)

Where: 

• 210,000 is the molar extinction coefficient (Ec) in cm^-1M^-1 of IgG at 280 nm.
• CF₂₈₀ is the correction factor for the effect of the fluorophore on absorbance at 280 nm.
• A_dye is the absorbance at maximum (λ_max) for the respective dye.

Table 1. Properties of Labeling Dyes found in the ReadiLink™ Rapid Antibody Labeling Kits.