AF594 Azide, ditriethylamine - BioReagent,≥90%(HPLC) , CAS No.A598145

CAS: A598145 Cat. No.: A598145 Molecular Weight: 1051.37

GRADE & PURITY BioReagent, ≥90%(HPLC)

Synonyms

Azide AF594 | AF 594 Azide | AF594 Azide, bis(triethylammonium salt)

Storage

Protected from light,Store at -20°C

Shipped In

Ice chest + Ice pads

Application

In Vivo Imaging, Protein/Antibody Labeling

Overview

Azide derivatives of AF fluorescent dyes react with alkynes via copper-catalyzed azide-alkyne cycloaddition (CuAAC). This click chemistry is increasingly used in a variety of biological applications. AF dye azides share the same chemical structure as AF dye azides and can react with terminal alkynes through CuAAC. Besides serving as extremely bright and photostable fluorophores for use with flow cytometry, microscopy, and HCS (High Content Screening), they also function as bioorthogonal or biologically unique haptens in applications requiring signal amplification.

Protocol for Labeling Alkyne-Modified Biomolecules with Fluorescent Azide Dyes

I. Labeling Oligonucleotides with Fluorescent Azide Dyes

1. Prepare the following stock solutions:
   • 200 mM THPTA (Tris(3-hydroxypropyltriazolylmethyl)amine) aqueous solution
   • 100 mM CuSO₄ aqueous solution
   • Alkyne-modified oligonucleotide aqueous solution (as concentrated as possible, e.g., >10 mg/mL)
   • 100 mM sodium ascorbate aqueous solution
   • 10 mM fluorescent azide dye in DMSO or water.
2. Mix CuSO₄ and THPTA at a 1:2 ratio, vortex for several minutes. The working solution is stable for weeks when frozen.
3. Add an excess of fluorescent azide dye to the alkyne-modified oligonucleotide solution (molar ratio: 2–5 eq.).
4. Add 5 eq. of the THPTA/CuSO₄ working solution (from Step 2).
5. Add 10–30 eq. of sodium ascorbate.
6. Stir, vortex, or shake the reaction mixture at room temperature for 30–60 minutes.
7. Purify the oligonucleotide by ethanol precipitation or your method of choice (e.g., HPLC).

II. Labeling Peptides with Fluorescent Azide Dyes

1. Prepare the following stock solutions:
   • 200 mM THPTA ligand aqueous solution
   • 100 mM CuSO₄ aqueous solution
   • Alkyne-modified peptide in water or DMF (as concentrated as possible to >10 mg/mL, depending on peptide solubility)
   • 100 mM sodium ascorbate aqueous solution
   • 10 mM fluorescent azide dye in DMSO or water.
2. Incubate CuSO₄ and THPTA ligand at a 1:2 ratio for several minutes. The solution is stable for weeks when frozen.
3. Add an excess of fluorescent azide dye to the alkyne-modified peptide solution (molar ratio: 5–10 eq.).
4. Add 5–10 eq. of THPTA/CuSO₄.
5. Add 10–20 eq. of sodium ascorbate.
6. Stir, vortex, or shake the reaction mixture at room temperature for 30–60 minutes.
7. Purify the desired peptide by HPLC.

III. Labeling Small-Molecule Organic Alkyne Compounds with Fluorescent Azide Dyes

1. Prepare the following stock solutions:
   • 200 mM THPTA ligand aqueous solution
   • 100 mM CuSO₄ aqueous solution
   • Alkyne compound in water or DMF (as concentrated as possible to >10 mg/mL, depending on compound solubility)
   • 100 mM sodium ascorbate aqueous solution
   • 10 mM fluorescent azide dye in DMSO or water.
2. Incubate CuSO₄ and THPTA ligand at a 1:2 ratio for several minutes. The solution is stable for weeks when frozen.
3. Add an excess of fluorescent azide dye to the alkyne solution (molar ratio: 5–10 eq.).
4. Add 25 eq. of THPTA/CuSO₄.
5. Add 50 eq. of sodium ascorbate.
6. Stir the reaction mixture at room temperature for 30–60 minutes.
7. Purify the desired molecule by chromatography or other methods.

IV. Labeling Biopolymers with Fluorescent Azide Dyes

1. Prepare the following stock solutions:
   • 200 mM THPTA ligand aqueous solution
   • 100 mM CuSO₄ aqueous solution
   • Alkyne-modified biopolymer aqueous solution (as concentrated as possible, e.g., >5 mg/mL)
   • 100 mM sodium ascorbate aqueous solution
   • 10 mM fluorescent azide dye in DMSO or water.
2. Incubate CuSO₄ and THPTA ligand at a 1:2 ratio for several minutes. The solution is stable for weeks when frozen.
3. Add an excess of fluorescent azide dye to the alkyne-modified biopolymer solution (loading ratio: 5–20 fluorescent azide dyes per alkyne).
4. Add 5 eq. of THPTA/CuSO₄ relative to the molar amount of the fluorescent azide dye.
5. Add 10 eq. of sodium ascorbate relative to the molar amount of the fluorescent azide dye.
6. Stir, vortex, or shake the reaction mixture at room temperature for 30–60 minutes.
7. Purify the desired molecule by gel filtration or dialysis.

V. Labeling Cells, Cell Lysates, or Biological Samples with Fluorescent Azide Dyes

1. Prepare the following click reaction solutions:
   • 100 mM THPTA ligand in aqueous buffer or water
   • 20 mM CuSO₄ aqueous solution
   • 300 mM sodium ascorbate aqueous solution
   • 2.5 mM alkyne or azide labeling reagent in water or DMSO.
2. For each azide- or alkyne-modified cell or cell lysate sample, add the following reagents to a 1.5 mL microcentrifuge tube and briefly vortex to mix: 50 μL cell or cell lysate sample, 50 μL PBS buffer, 50 μL 5 mM corresponding fluorescent azide dye (or dye alkyne) detection reagent in DMSO or water.
3. Add 10 μL of 100 mM THPTA solution and briefly vortex to mix.
4. Add 10 μL of 20 mM CuSO₄ solution and briefly vortex to mix.
5. Add 10 μL of 300 mM sodium ascorbate solution to initiate the click reaction, then briefly vortex to mix.
6. Protect the reaction from light and incubate at room temperature for 30 minutes.
7. The cells or cell lysates are now click-labeled and ready for downstream processing and analysis.

Specifications

Synonyms

Azide AF594 | AF 594 Azide | AF594 Azide, bis(triethylammonium salt)

Specifications & Purity

BioReagent, ≥90%(HPLC)

Stability And Storage

Store at -20℃ long term (1 year). Store in the dark.

Storage

Protected from light, Store at -20°C

Shipped In

Ice chest + Ice pads

This product requires cold chain shipping. Ground and other economy services are not available.

Grade

BioReagent

Purity

≥90%(HPLC)

Names and Identifiers

• Molecular Weight: 1051.37