DyLight fluorophores in cell analysis

Choose an appropriate fluorescent labeling reagent or probe.

Thermo Scientific DyLight Fluorescent Dyes are a complete family of high-intensity, photostable fluorescent tags for labeling antibodies and other molecular probes.

The DyLight Fluors are available as reactive labeling agents and as conjugates of secondary antibodies, biotin-binding proteins and molecular weight markers for use in fluorescence microscopy, flow cytometry, ELISA, high-content screening and other array platforms.

Pierce Protein Methods

Properties of DyLight Fluorophores

DyLight Fluors have absorption maxima ranging from 350nm to 777nm, covering the entire visible light spectrum and several key near-infared and infrared wavelengths. The absorption and emission properties of the DyLight Fluors match the output (excitation) and detection wavelengths of common fluorescence instrumentation.

The DyLight Dyes exhibit higher fluorescence intensity and photostability than Alexa Fluor, CyDye and LI-COR Dyes in many applications and remain highly fluorescent over a broad pH range (pH 4-9). Additionally, the water solubility of the DyLight Dyes allows a high dye-to-protein ratio to be achieved without causing precipitation of conjugates.

Emission spectra of DyLight dyes

Figure 1. Emission spectra of Thermo Scientific DyLight dyes. See table below for additional specifications and links to detailed information pages about each dye.
 

Spectral properties of DyLight fluorophores.

Each dye name and spectrum-image links to a dedicated information page that includes the detailed spectra, example data and literature references.

Emission Fluor Ex/Em† Spectra 円 Spectrally Similar Dyes
Blue DyLight 350 353/432 Description: Excitation and Emission Spectra of DyLight 350 Fluorophore 15K Alexa Fluor 350, AMCA
Blue DyLight 405 400/420 Description: Excitation and Emission Spectra of DyLight 405 Fluorophore 30K Alexa Fluor 405, Cascade Blue
Green DyLight 488 493/518 Description: Excitation and Emission Spectra of DyLight 488 Fluorophore 70K Alexa Fluor 488, fluorescein, FITC
Yellow DyLight 550 562/576 Description: Excitation and Emission Spectra of DyLight 550 Fluorophore 150K Alexa Fluor 546, Alexa Fluor 555, Cy3,TRITC
Red DyLight 594 593/618 Description: Excitation and Emission Spectra of DyLight 594 Fluorophore 80K Alexa Fluor 594, Texas Red
Red DyLight 633 638/658 Description: Excitation and Emission Spectra of DyLight 633 Fluorophore 170K Alexa Fluor 633
Red DyLight 650 652/672 Description: Excitation and emission spectra for DyLight 650 250K Alexa Fluor 647, Cy5
Near IR DyLight 680 692/712 Description: Excitation and Emission Spectra of DyLight 680 Fluorophore 140K Alexa Fluor 680, Cy5.5
Near IR
DyLight 755 754/776 Description: Excitation and Emission Spectra of DyLight 750 Fluorophore 220K Alexa Fluor 750
Infrared DyLight 800 777/794 Description: Excitation and Emission Spectra of DyLight 800 Fluorophore 270K IRDye 800

†Excitation and emission maxima in nanometers (+/- 4nm) in phosphate-buffered saline (PBS)
††Molar extinction coefficient (M-1 cm-1) at the absorption maximum>

DyLight Reactive Dyes and Labeling Kits

The DyLight Fluorescent Dyes are available in both NHS ester-activated and maleimide-activated forms, which provide for efficient and specific labeling of primary amines and sulfhydryl groups, respectively. Several DyLight Fluors are also available with azide-reactive phosphine groups for detection and labeling using Staudinger ligation chemistry. Certain DyLight Fluors are also available as free acids for use as non-reactive negative controls.

The amine-reactive DyLight Fluors are available as both stand-alone reagents and complete kits optimized for milligram- and microgram-scale antibody or general protein labeling reactions. The DyLight Fluor Antibody Labeling Kits include the amine-reactive DyLight Fluors, buffers and special columns for easy clean-up of the labeling reaction and recovery of the labeled protein. The Dye Removal Columns are also available separately.
 

Thermo Scientific DyLight Reactive Dyes

Emission Fluor Available Derivatives
NHS Ester Maleimide Phosphine Free Acid†
Blue DyLight 350 Yes Yes    
Blue DyLight 405 Yes Yes    
Green DyLight 488 Yes Yes Yes Yes
Yellow DyLight 550 Yes Yes Yes  
Red DyLight 594 Yes Yes    
Red DyLight 633 Yes Yes    
Red DyLight 650 Yes Yes Yes  
Near IR DyLight 680 Yes Yes    
Near IR DyLight 747       Yes
Near IR DyLight 755 Yes Yes    
Infrared DyLight 800 Yes Yes   Yes

†Free-acid forms of the DyLight Fluors are unreactive and used as negative controls.

DyLight Fluorescent Probes (Conjugates)

Pre-conjugated secondary antibodies and biotin-binding proteins of DyLight Fluors make excellent secondary detection reagents for fluorescent assays. Our DyLight Fluor Conjugates is optimally labeled with the highest dye-to-protein ratio (F/P ratio) that does not cause quenching of the fluorescent signal or problems with conjugate solubility.

  • Secondary Antibody Conjugates — anti-mouse IgG and anti-rabbit IgG secondary antibodies conjugated to DyLight Fluors make a variety of immunodetection, immunoassay and cellular imaging procedures accessible to fluorescence measurement and multiplexing experimental strategies. 
  • Highly Cross-Adsorbed Secondary Antibody Conjugates — these forms of secondary antibodies are specially purified to minimize binding to off-target immunoglobulins, making them ideal for multiplex immunofluorescence assays where cross-reactivity is a concern. 
  • Streptavidin Conjugates — streptavidin biotin-binding protein conjugated to DyLight Fluors enables detection of biotinylated primary antibodies and other biotin-labeled targets for fluorescence methods.
  • NeutrAvidin Conjugates — Thermo Scientific NeutrAvidin Biotin-Binding Protein is an economical and equally high-performance alternative to streptavidin for biotin detection methods.
     

Assay Platforms Based on DyLight Fluor Conjugates

DyLight Fluor conjugated secondary antibodies and other molecular probes are the basis for a variety of optimized assay platforms for multiplex and high-content analysis of cellular pathway targets.

In-Cell ELISA Kits— The Thermo Scientific Near Infrared In-Cell ELISA Kits use pairs of DyLight 680 and DyLight 800 conjugated secondary antibodies to quantify expression of specific targets in whole cells using an ELISA format.

  • A generic In-Cell ELISA Kit allows users to develop a target-specific assay using their own primary antibodies. 
  • Target-specific In-Cell ELISA Kits include optimized primary antibodies for particular proteins of research interest (EGFR, ERK, STAT3, etc.).

High Content Screening Assays— Thermo Scientific High Content Reagent-Based Assays are multiplex fluorescent assays for cellular imaging and analysis. Each one of these many assays uses DyLight Fluor Antibodies and other fluorescent probes to detect the cellular expression and translocation of specific proteins and cell structural parameters. When used in combination with appropriate imaging equipment and analysis software, these assays provide extremely powerful strategies for examining cellular responses.