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Chromeo™ 546

effective red fluorescent dye for bioanalysis

Chromeo™ 546 is a bright, red fluorescent label that replaces Cy3*, Alexa Fluor 546*, DyLight 549* and other dyes excitable within a wavelength range of 530-555 nm. It exhibits superior luminescence properties, a broad Stokes shift, stability towards photobleaching and pH, and low cell toxicity. Chromeo 546 is compatible with various excitation sources including mercury arc, tungsten and xenon arc lamps, the 544 nm line of the Helium-Neon laser and the 532 nm green laser line.

Why use Chromeo™ 546?

  • Photostability – enables multiple exposures and increased exposure time
  • Bright fluorescence – sensitive detection
  • Low background
  • pH insensitive
  • Easy to use – no harsh chemicals required for conjugation
  • Narrow emission and absorption bands – enables multi-color experiments

Reactive fluorescent Chromeo 546 serves as a bright label for antibodies and other biomolecules, enabling detection in immunofluorescence, high content screening, ELISA and FRET applications, as well as in flow cytometry. To provide you with flexibility in labeling your antibodies and proteins, Chromeo 546 is offered in a variety of chemistries. For greater convenience, Chromeo 546 can be purchased in our easy-to-use Fluorescent Antibody Labeling Kits, and is also offered as optimized Fluorescent Secondary Antibody Conjugates for enhanced detection.

To ensure that you get the best possible results in your immunofluorescent staining, we recommend the use of Chromeo 546 together with our complete line of MAX Stain™ Immunofluorescence Tools, as those components have been formulated to optimize the performance of all Fluorescent Chromeo™ Dyes.

 
Name Format Cat No. Price  
Chromeo™ 546 Azide 1 mg 15284 $240 Buy Now
5 x 1 mg 15285 $925 Buy Now
Chromeo™ 546 Alkyne 1 mg 15294 $240 Buy Now
5 x 1 mg 15295 $925 Buy Now
Chromeo™ 546 Carboxylic Acid 1 mg 15210 $115 Buy Now
5 x 1 mg 16210 $445 Buy Now
Chromeo™ 546 NHS-Ester 1 mg 15211 $230 Buy Now
5 x 1 mg 16211 $865 Buy Now
Chromeo™ 546 Streptavidin 1 mg 15213 $210 Buy Now
5 x 1 mg 16213 $825 Buy Now
 
Chromeo 546 Fluorescent Dye Spectra
 
Figure 1: Absorption and emission spectra of Chromeo™ 546.

 

Dye Absorption Emission ε L/(mol-cm) Stokes shift
Chromeo™ 546 545 nm 561 nm 98,800 16 nm
Table 1: Properties of Chromeo™ 546.
 

Chromeo™ 546 is a bright fluorescent dye that can be excited between 530-555 nm. The dye is very stable over a broad pH range and in combination the MAX Stain™ reagents it shows high fluorescence intensity and photostability. Chromeo 546 and Chromeo 546 Fluorescent Secondary Antibody Conjugates are a good choice to replace dyes like Cy3*, Alexa Fluor 546*, DyLight 549* and their conjugates

 
HeLa cells stained by alpha Tubulin mAb and Chromeo 546 Fluorescent Secondary Antibody
 
Figure 1: Chromeo 546 staining in HeLa cells.

HeLa cells were stained with alpha Tubulin mouse mAb (Clone 5-B-1-2) and Chromeo 546 Goat anti-mouse IgG (Catalog No. 15033).

To evaluate the photostability of Chromeo 546 conjugates in immunofluorescence experiments, tubulin was stained in HeLa cells and the intensity of it and various other secondary antibodies was monitored over a period of 80 seconds. The comparison with ATTO 550, Cy3, Alexa 546 and DyLight 549 in Figure 2 shows that most of the conjugates exhibit similar resistance to photobleaching when the samples were prepared under the same conditions reagents with MAX Stain reagents. The Alexa 546 conjugates perform poorly and exhibit photobleaching when prepared using MAX Stain reagents, though these same reagents prevent fading with the other dyes we have tested.

 
Photobleaching analysis of dyes excitable in the 530-550 nm range
 
Figure 2: Photobleaching analysis of various dyes excitable in the 530-550 nm range while using MAX Stain reagents.
 

All Chromeo dyes have been evaluated in a variety of fluorescent assays and analyzed with different instruments, such fluorescent plate readers and Widefield/Structured Illumination & Confocal microscopes. Figure 3 shows a comparison of three different dye conjugates, Chromeo 546 Goat anti-mouse IgG, Alexa 546 anti-mouse IgG and DyLight 549 anti-mouse IgG, when excited using the 544 nm line of a HeNe laser. The figure demonstrates the quality of the Chromeo 546 conjugated secondary antibodies.

 
alpha Tubulin staining by Chromeo 546, Alexa 546 and DyLight 549 fluorescent secondary antibody conjugates
 
Figure 3: Tubulin staining in HeLa cells, analyzed by confocal microscopy using the 544 nm line of the HeNe laser.

HeLa cells were stained with alpha Tubulin mouse mAb (Clone 5-B-1-2) and visualized with either Chromeo 546 Goat anti-mouse IgG (Catalog No. 15033), Alexa 546 anti-mouse IgG or DyLight 549 anti-mouse IgG.

 

Figure 4 shows the confocal microscopy picture of the Chromeo 546 conjugate with an inset at a greater enlargement. The ability to zoom in closely and see the tubulin fibers clearly demonstrates the dye's brightness and low background in immunofluorescent staining. This makes Chromeo 546 and its Fluorescent Secondary Antibody Conjugates excellent tools for use in fluorescent microscopy experiments.

 
Enlarged image of alpha Tubulin mAb and Chromeo 546 Fluorescent Secondary Antibody immunofluorescent staining
 
Figure 4: Tubulin staining in HeLa cells, analyzed by confocal microscopy using the 544 nm line of the HeNe laser.

HeLa cells were stained with alpha Tubulin mouse mAb (Clone 5-B-1-2) and Chromeo 546 Goat anti-mouse IgG (Catalog No. 15033).

 
Chromeo 546 secondary antibody used to stain EGFR in urothel cells
 
Figure 5: EGFR staining with Chromeo 546 secondary antibody.

Chromeo 546 Goat anti-mouse IgG (Catalog No. 15033) was used to stain EGFR in an urothel cell line. This data was generously provided by Dr. Brockhoff, Institut for Pathology, University Regensburg, Germany.

Chromeo 546 has been shown to work in several different types of fluorescence microscopy. In addition to classical confocal or widefield applications, Chromeo 546 and its Fluorescent Secondary Antibody Conjugates have been tested in super-resolution microscopy. For GSDIM (Ground State Depletion with Individual Molecule return) microscopy using the Leica GSD SR microscope, Active Motif’s fluorescent Chromeo 546 dye and conjugated secondary antibodies have been tested and recommended by Leica Microsystems. The fluorescent properties of Chromeo 546 dye and secondaries meet the specifications required to perform GSDIM microscopy, as shown below by tubulin staining (Figure 6).

Comparison of conventional widefield microscopy and GSDIM microscopy using Chromeo 546 Goat anti-mouse IgG secondary antibody
Figure 6: Chromeo 546 antibody conjugates in widefield and GSDIM microscopy.

Tubulin was stained with a primary monoclonal mouse antibody and with Chromeo 546 Goat anti-mouse IgG (Catalog No. 15033) secondary antibody. The widefield (left) and GSDIM (right) images are courtesy of Leica Microsystems, Germany.

The following selected publications cite the use of and/or provide additional information about Chromeo 546:

The Chromeo 546 Antibody Labeling Kit has been cited in the following publications:

Contents & Storage

Chromeo™ 546 NHS-Ester and Chromeo™ 546 Carboxylic Acid are supplied lyophilized and should be stored at the recommended temperature in the dark. The Chromeo™ 546 Streptavidin conjugate is supplied as a 1 mg/ml solution in PBS and should be stored at 4°C in the dark to ensure photostability.

All products are guaranteed 6 months when stored properly.

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