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Labeling Azide-Modified Biomolecules with Fluorescent Dye Alkynes
Labeling Oligonucleotides with Dye Alkynes
  1. Prepare the following stock solutions:
    • 200 mM THPTA [tris(3- hydroxypropyltriazolylmethyl)amine)] in water
    • 100 mM CuSO4 in water
    • Azide-modified oligo in water (as concentrated as possible, e.g., >10 mg/ml)
    • 100 mM sodium ascorbate in water
    • 10 mM dye alkyne in DMSO or water (see our website for recommended solvent)
  2. Mix and vortex well CuSO4 with THPTA in a 1:2 ratio for several minutes before the reaction. This working solution is stable for several weeks when frozen.
  3. To the azide-modified oligo solution, add an excess of dye alkyne (2-5 equivalents by molar ratio).
  4. Add 5 equivalents of THPTA/CuSO4working solution (from Step 1)
  5. Add 10-30 equivalents of sodium
  6. Stir, vortex or shake the reaction mixture at room temperature for 30-60 minutes.
  7. Ethanol-precipitate the oligo or purify it by your desired method (e.g., HPLC).
Labeling Peptides with Dye Alkynes
  1. Prepare the following stock solutions:
    • 200 mM THPTA ligand in water
    • 100 mM CuSO4 in water
    • Azide-modified peptide in water or DMF (depending on your peptide solubility, >10 mg/ml if possible)
    • 100 mM sodium ascorbate in water
    • 10 mM dye alkyne in DMSO or water (see our website for recommended solvent)
  2. Incubate CuSO4 with THPTA ligand in a 1:2 ratio several minutes before the reaction. This solution is stable for several weeks when frozen.
  3. To the azide-modified peptide solution, add an excess of dye alkyne (5-10 equivalents by molar ratio).
  4. Add 5-10 equivalents of THPTA/CuSO4.
  5. Add 10-20 equivalents of sodium.
  6. Stir, vortex or shake the reaction mixture at room temperature for 30-60 minutes.
  7. Purify your desired peptide by HPLC.
Labeling Small Organic Alkyne Molecules with Dye Alkynes
  1. Prepare the following stock solutions:
    • 200 mM THPTA ligand in water
    • 100 mM CuSO4 in water
    • Alkyne compound in water or DMF (depending on your compound solubility, >10 mg/ml if possible,)
    • 100 mM sodium ascorbate in water
    • 10 mM dye alkyne in DMSO or water (see our website for recommended solvent).
  2. Incubate CuSO4 with THPTA ligand in a 1:2 ratio several minutes before the reaction. This solution is stable for several weeks when frozen.
  3. To the alkyne solution, add an excess of dye alkyne (5-10 equivalents by molar ratio).
  4. Add 25 equivalents of THPTA/CuSO4.
  5. Add 50 equivalents of sodium
  6. Stir the reaction mixture at room temperature for 30-60 minutes.
  7. Purify your desired molecule by chromatography or other methods.
Labeling Biopolymers with Dye Alkynes
  1. Prepare the following stock solutions:
    • 200 mM THPTA ligand in water
    • 100 mM CuSO4 in water
    • Azide-modified biopolymer in water (as concentrated as possible, e.g., >5 mg/ml)
    • 100 mM sodium ascorbate in water
    • 10 mM dye alkyne in DMSO or water (see our website for recommended solvent).
  2. Incubate CuSO4 with THPTA ligand in a 1:2 ratio several minutes before the reaction. This solution is stable for several weeks when frozen.
  3. To the azide-modified biopolymer solution, add an excess of dye alkyne (Loading ratio: 5-20 dye alkyne/alkyne).
  4. Add 5 molar equivalents (referenced to dye alkyne) of THPTA/CuSO4.
  5. Add 10 equivalents of sodium ascorbate (referenced to dye alkyne).
  6. Stir, vortex or shake the reaction mixture at room temperature for 30-60 minutes.
  7. Purify your desired molecule by gel filtration or dialysis.
Document: 02.0117.180413r1
Last updated Mon Oct 13 2025
Labeling Azide-Modified Biomolecules with Fluorescent Dye Alkynes
Labeling Oligonucleotides with Dye Alkynes
Labeling Peptides with Dye Alkynes
Labeling Small Organic Alkyne Molecules with Dye Alkynes
Labeling Biopolymers with Dye Alkynes