LumiDye™ AF700 NHS Ester is a bright and photostable near-IR dye that can be excited with a xenon-arc lamp, far-red diode lasers, or dye pumped lasers operating in the 675–700 nm range. Used for stable signal generation in imaging and flow cytometry, LumiDye™ AF700 dye is water soluble and pH-insensitive from pH 4 to pH 10. NHS esters can be used to label to the primary amines (R-NH2) of proteins, amine-modified oligonucleotides, and other amine-containing molecules. The resulting AF700-conjugate will exhibit brighter fluorescence and greater photostability than the conjugates of other spectrally similar fluorophores. In addition to reactive dye formulations, we offer AF700 dye conjugated to a variety of antibodies, peptides, proteins, tracers, and amplification substrates optimized for cellular labeling and detection.
Detailed information about this LumiDye™ AF700 NHS Ester:
Fluorophore label: LumiDye™ AF700 NHS Ester
Reactive group: NHS ester
Reactivity: Primary amines on proteins and ligands, amine-modified oligonucleotides
Ex/Em of the conjugate: 696/719 nm
Extinction coefficient: 19,000 cm-1M-1
Molecular weight: 1400
CF280: 0.07
Spectrum:
Typical Labeling Protocol
1. Dissolve ~10 mg of the protein in 1 mL of 0.1 M sodium bicarbonate buffer pH 8.3.
2. Preparing the Reactive Dye Solution: Immediately before use, dissolve the Reactive dye in high-quality, anhydrous dimethylsulfoxide (DMSO) or dimethylformamide (DMF) at a final concentration of 10 mg/mL. Once reconstituted, use the reactive dye solution immediately.
3. While stirring or vortexing the protein solution (step 1), slowly add 50–100 μL of the reactive dye solution.
4. Incubate the reaction for 1 hour at room temperature with continuous stirring.
5. Optional: Stop the reaction by adding 0.1 mL of freshly prepared 1.5 M hydroxylamine, pH 8.5, and incubate the hydroxylamine-containing reaction for one hour at room temperature.
6. Separating the conjugate from unreacted labeling reagent by dialysis or gel filtration media with an appropriate molecular weight cutoff.
7. Determining the Degree of Labeling: Measure the absorbance of the protein–dye conjugate at 280 nm (A₂₈₀) and at the λmax for the dye (Aₘₐₓ).
8. The following formula can be used to calculate the protein concentration:
Protein concentration (mg/mL) = (A280 - CF₂₈₀ × Amax) ×MW/εprotein
9. The following formula can be used to calculate the degree of labeling:
DOL = (Amax × MW) / (Protein concentration× εdye)
where MW = the molecular weight of the protein, εdye = the extinction coefficient of the
dye at its absorbance maximum, and the protein concentration is in mg/mL.
10. Add 0.05–0.2% Proclin 300 or 0.05% sodium azide, along with a protein stabilizer (such as 0.1% BSA), to the labeled protein.
