AbBy Fluor™ NHS Esters

PROTOCOL
Important: This protocol is optimized for labeling 10 mg of an IgG antibody. It can be scaled up or down as needed, provided the molar ratios of reagents remain consistent. Since the reactivity between different proteins and AbBy Fluor™ NHS esters can vary significantly, it is recommended to test three different molar ratios of the reactive reagent to protein to achieve the best results for your specific protein.

1.1 Dissolve ~10 mg of the protein in 1 mL of 0.1 M sodium bicarbonate buffer. The protein concentration in the reaction should usually be 5-10 mg/mL. Concentrations lower than 2 mg/mL will greatly decrease the efficiency of the reaction.

Protein solutions must be free from amine-containing substances such as Tris, glycine, ammonium ions, or stabilizing proteins like bovine serum albumin. Antibodies dissolved in buffers containing amines can be dialyzed against PBS to remove these contaminants. To achieve the desired pH for the reaction, add 0.1 mL of 1 M sodium bicarbonate buffer per mL of antibody solution. Note that sodium azide interferes with the conjugation reaction, so dialysis is recommended to remove it if necessary.

1.2 Dissolve the AbBy Fluor™ NHS ester in DMF or DMSO to make 10 mg/ml concentration. For a typical reaction, dissolve 1 mg of AbBy Fluor™ NHS ester in 100 μL of DMF or DMSO. Prepare the dye solution immediately before starting the reaction, as reactive compounds are not very stable in solution. Briefly sonicate or vortex to ensure complete dissolution.

1.3 While stirring or vortexing the protein solution (step 1.1), slowly add 50-100 μL of the AbBy Fluor™ NHS ester solution (step 1.2).

1.4 Incubate the reaction for 1 hour at room temperature with continuous stirring.

1.5 Equilibrate a 10 × 300 mm gel filtration column (Sephadex® G-25, BioGel® P-30, or equivalent) with PBS.

1.6 Separate the conjugate on the gel filtration column.

1.7 Store the conjugates under the same conditions used for the parent protein. For storage in solution at 2-8°C, add suitable preservative if needed.

Determining the Degree of Labeling

2.1 Measure the absorbance of the protein-dye conjugate at 280 nm (A₂₈₀) and at the λ_max for the dye (A_max). Dilute the protein-dye conjugate to approximately 0.1 mg/mL, preparing only the amount needed for the measurement. Refer to Table 2 for the λ_max values of AbBy Fluor™ dyes.

2.2 Calculate the degree of labeling (DOL):
DOL = (A_max ×ε_protein)/(ε_dye x (A₂₈₀-CF₂₈₀×A_max))
Note: ε of IgG is ~210,000 M^-1 cm^-1 and CF₂₈₀ values for AbBy Fluor™ dyes are given in the Table 2.