SureLight 488 Labeling Protocol

1. Antibody solutions must be free of any amine-containing substances such as Tris, glycine, ammonium ions, or stabilizing proteins such as bovine serum albumin. You can dialyze antibodies that have been previously dissolved in buffers containing amines against 100 mM sodium bicarbonate, pH 8.3 as a reaction buffer. Antibodies that are currently in non-amine containing buffers such as phosphate buffered saline (PBS), can often be adjusted to the desired pH for the reaction by adding 0.1 mL of 1 M sodium bicarbonate buffer (pH 8.3) for each mL of antibody solution.
2. Dissolve the 1 mg or 100 ug vial of Surelight 488 in dry DMF or DMSO at 10 mg/mL. For a typical reaction, dissolve 1 mg of dye in 0.1 mL of solvent. Dissolve the dye immediately before starting the reaction as the reactive compounds are not very stable in solution long term. Briefly vortex to ensure thorough dissolution.
3. Refer to labeling table to get the preferred labeling ratio. We recommend aiming for 4-6 dyes per antibody.
4. Add, the appropriate volume, mix thoroughly by vortexing, and incubate the reaction for 1 hour at room temperature with continuous stirring or on an orbital rocker.
5. After 1h purify the conjugate by desalting (Zeba or G25 resin) or dialyze exhaustively into PBS.
6. After purification, measure the absorbance of the conjugate solution at 280 nm and 494 nm (A280 and A494) in a cuvette with a 1 cm pathlength. Note: dilution of the sample may be necessary.
7. Calculate the concentration of protein in the sample.
   • The protein concentration [M_protein] = [A280 – (A494 ? CF)] ? e_protein ?x dilution factor
   • where CF = the correction factor at 280nm for the dye = 0.1
   • e_protein = the molar extinction coefficient for the protein at 280nm. For IgG this typically = 228000M^-1cm
   • Non-IgG proteins will likely have significantly different molar extinction coefficients.
8. Calculate the degree of labeling (DOL).
   • DOL (in moles dye per mole protein) = (A494 ? e_dye x dilution factor)/ [M_protein]
   • where e_dye = the molar extinction coefficient for the dye at 494nm = 71000
9. Store the labeled protein in PBS, pH 7.2 + 2 mM sodium azide at 2?6?C, and protected from light. If the final concentration of purified protein conjugate is less than 1 mg/mL, add bovine serum albumin (BSA) or another stabilizing protein to 1?10 mg/mL.