Conjugation Protocol for Amine Reactive CoraFluor™ Reagents

Introduction

Amine reactive CoraFluor™ reagents contain pentafluorophenyl esters (PFP esters) which can be conjugated to (non-protonated) aliphatic amine groups. The primary reactive species for protein amine-conjugation are the ε-amino groups of lysine residues. To avoid protonating these groups, it is important to perform the reaction at a slightly basic pH. In addition, buffers containing primary amines should be avoided, since they will compete for conjugation with the PFP ester.

Please note that PFP esters can be moisture sensitive, so handle accordingly. Where possible, handle and store CoraFluor™ reagents in the dark.

Conjugation Protocol

• Prepare a 100 μL aliquot of an antibody, protein or nanobody at a concentration of ≥1 mg/mL in reaction buffer (100 mM sodium carbonate buffer, pH 8.5) using a 0.5 mL, 7 kDa molecular weight cutoff (MWCO) Zeba™ spin desalting column (Thermo Fisher 89882) according to the manufacturer’s protocol.
• Add the antibody/protein/nanobody solution to the amine reactive CoraFluor™ to achieve a molar ratio of ~5–15× CoraFluor™ to antibody/protein, or 4–5× CoraFluor™ to nanobody.

If performing multiple conjugations, reconstitute CoraFluor™ in 2.5 mM dry DMSO or DMAc and perform conjugation reactions with a final DMSO or DMAc content <10%. The molar equivalents of CoraFluor™ can be adjusted accordingly depending on size of the protein and desired degree of labeling.

• Briefly vortex the reaction mixture and incubate at room temperature for 1 h.
• Remove organic solvent and unreacted PFP ester complex by buffer exchange into desired storage buffer (e.g. 50 mM sodium phosphate buffer, pH 7.4, with 150 mM NaCl and 0.05% (vol/vol) TWEEN^®-20) using a 0.5 mL, 7 kDa MWCO Zeba™ spin desalting column, according to the manufacturer’s protocol.
• Determine concentration and degree of labeling (DOL) using the below calculations.

Degree of Labeling Calculation

• Determine the corrected absorbance at 280 nm value (A_280,corr) of the antibody/nanobody/protein conjugate by measuring A₂₈₀ and A₃₄₀ using:

          A_280,corr = A₂₈₀ - (A₃₄₀ x c.f.)

c.f. is the correction factor for the terbium complex contribution to A₂₈₀ and is equal to 0.157.

• Determine the concentration of antibody/protein/nanobody conjugate, c_ab (in M) using:

  cab =	A280,corr	x b
  	εab

where ε_ab is the antibody/protein/nanobody extinction coefficient at A₂₈₀ and b is the path length in centimeters.

• Determine the concentration of covalently bound terbium complex, c_Tb (in M) using:

  cTb =	A340	x b
  	εTb

where ε_Tb is the complex extinction coefficient at A₃₄₀, equal to 22,000 M⁻¹ cm⁻¹, and b is the path length in centimeters.

• Calculate the degree of labeling (DOL) using:

  DOL =	cTb	x b
  	cab

TWEEN^® is a registered trademark of Croda International PLC

Zeba™ is a registered trademark of Thermo Fisher Scientific

For more information on preparing stock solutions and reconstitution, please see our Molarity Calculator and Reconstitution Calculator.