Fluorescent Dye Selection Guide | AF488, AF568, AF647, Cy3, Cy5 & Cy7

FLUORESCENT LABELING GUIDE

Fluorescent Dye Selection Guide:
AF vs Cyanine vs BDP®

From AF488, AF568, and AF647 to Cyanine3, Cyanine5, Cyanine7, and BDP® dyes: learn how to select a fluorophore based on spectral range, solubility, labeling chemistry, and experimental application.

Choosing a Fluorescent Dye Is More Than Choosing a Wavelength

Excitation and emission wavelengths are usually the first considerations when selecting a fluorescent dye. However, fluorophores operating in similar spectral regions can differ in brightness, photostability, hydrophilicity, and conjugation behavior.

These differences can become important in antibody labeling, protein conjugation, fluorescence microscopy, flow cytometry, multiplex imaging, and in vivo applications.

Quick Selection Guide

Green: AF488 / BDP® FL
Orange-Red: AF568 / Cyanine3 family
Far-Red: AF647 / Cyanine5 family
Near-Infrared: Cyanine7 family

Fluorescent Dyes by Spectral Region

Spectral Region Representative Dyes Typical Channel Common Applications
Green AF488, BDP® FL FITC / FAM region Microscopy, flow cytometry, general labeling
Orange Cyanine3, Cyanine3B, AF568 Cy3 / TRITC region Protein labeling, fluorescence microscopy
Far-Red AF647, Cyanine5, sulfo-Cyanine5 Cy5 / far-red region Antibody labeling, multiplex imaging
NIR Cyanine7, sulfo-Cyanine7 Near-infrared NIR and in vivo imaging

AF Fluorescent Dyes

AF dyes provide useful options for applications requiring bright, photostable fluorescence across several commonly used detection channels.

AF488
A green fluorophore suitable for common FITC-like detection channels, fluorescence microscopy, and flow cytometry.
AF568
A bright orange-red fluorophore useful for protein labeling, antibody conjugation, and fluorescence imaging.
AF647
A far-red fluorophore particularly useful for antibody labeling and multicolor fluorescence experiments.

Cyanine Fluorescent Dyes

Cyanine dyes cover a broad spectral range from visible fluorescence to near-infrared wavelengths, making the family useful for a wide variety of labeling and imaging applications.

Cyanine3 and Cyanine3B

Cyanine3-family dyes provide yellow-to-orange fluorescence and are commonly used for biomolecule labeling and fluorescence imaging.

Cyanine5

Cyanine5 is a widely used far-red fluorophore. Its longer-wavelength fluorescence makes it useful in multicolor experiments together with green and orange fluorophores.

Cyanine7

Cyanine7-family fluorophores operate in the near-infrared region and can be useful for NIR detection and in vivo fluorescence imaging.

Cyanine vs sulfo-Cyanine

One important consideration within the Cyanine family is whether to use a conventional Cyanine dye or a sulfo-Cyanine derivative.

Sulfonated Cyanine dyes generally provide improved hydrophilicity and water solubility, which can be beneficial for protein and antibody conjugation in aqueous biological systems.

Feature Cyanine sulfo-Cyanine
Water Solubility Varies by derivative Generally higher
Hydrophilicity Relatively lower Higher
Typical Use General fluorescent labeling Aqueous protein and antibody conjugation

BDP® Fluorescent Dyes

Lumiprobe BDP® dyes are borondipyrromethene-based fluorophores designed for fluorescence labeling and imaging applications where bright and photostable fluorescence is desirable.

BDP® FL, for example, operates in the green fluorescence region and can be detected using many systems configured for FAM/FITC-like fluorophores.

Choosing a Dye by Application

Application Dyes to Consider Why
General fluorescence labeling AF488 / BDP® FL Compatible with common green detection channels
Antibody labeling AF568 / AF647 / sulfo-Cyanine5 Bright signals and flexible spectral options
Multiplex imaging AF488 + AF568 + AF647 Separation across multiple spectral channels
Aqueous protein conjugation sulfo-Cyanine derivatives Improved hydrophilicity and water solubility
NIR / in vivo imaging Cyanine7 / sulfo-Cyanine7 Near-infrared fluorescence

Next, Choose the Reactive Group

Selecting the fluorophore is only the first step. You also need to select the appropriate reactive group for attaching the dye to your target.

NHS Ester
Primary amine labeling
Proteins and antibodies
Maleimide
Thiol labeling
Cysteine conjugation
Azide / DBCO
Copper-free click chemistry
Bioorthogonal labeling
TCO / Tetrazine
Fast IEDDA chemistry
Rapid bioorthogonal conjugation

A Simple Fluorophore Selection Workflow

STEP 1. Check your instrument's excitation and emission channels.
STEP 2. Select green, orange, far-red, or NIR fluorescence.
STEP 3. Choose AF, Cyanine, sulfo-Cyanine, or BDP®.
STEP 4. Identify the functional group on your target biomolecule.
STEP 5. Select NHS ester, maleimide, DBCO, azide, TCO, or tetrazine chemistry.

Lumiprobe Fluorescent Labeling Reagents

Lumiprobe offers fluorescent dyes across multiple spectral regions together with a broad selection of reactive groups for biomolecule labeling.

  • AF fluorescent dyes
  • Cyanine and sulfo-Cyanine dyes
  • BDP® fluorescent dyes
  • NHS ester labeling reagents
  • Maleimide derivatives
  • Azide / DBCO click chemistry reagents
  • TCO / tetrazine bioorthogonal reagents
DANA BIOSCIENCE · FLUORESCENT LABELING REAGENTS

Not Sure Which Fluorescent Dye to Choose?

Tell us your instrument's laser/filter configuration, desired fluorescence channel, target biomolecule, and conjugation chemistry. Dana Bioscience can help identify suitable Lumiprobe reagents for your workflow.

Send us your application requirements or catalog numbers for pricing and availability.

For research use only. Spectral properties, instrument compatibility, and labeling conditions should be verified using the specifications of the individual reagent.
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