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.
|Chromeo™ 546 Azide||1 mg||15284||$220||Buy Now|
|5 mg||15285||$880||Buy Now|
|Chromeo™ 546 Alkyne||1 mg||15294||$220||Buy Now|
|5 mg||15295||$880||Buy Now|
|Chromeo™ 546 Carboxylic Acid||1 mg||15210||$100||Buy Now|
|5 mg||16210||$400||Buy Now|
|Chromeo™ 546 NHS-Ester||1 mg||15211||$210||Buy Now|
|5 mg||16211||$820||Buy Now|
|Chromeo™ 546 Biotin||1 mg||15212||$175||Buy Now|
|5 mg||16212||$700||Buy Now|
|Chromeo™ 546 Streptavidin||1 mg||15213||$195||Buy Now|
|5 mg||16213||$780||Buy Now|
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
Figure 1: Chromeo 546 staining in HeLa cells.
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.
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.
Figure 3: Tubulin staining in HeLa cells, analyzed by confocal microscopy using the 544 nm line of the HeNe laser.
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.
Figure 4: Tubulin staining in HeLa cells, analyzed by confocal microscopy using the 544 nm line of the HeNe laser.
Figure 5: EGFR staining with Chromeo 546 secondary antibody.
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).
Figure 6: Chromeo 546 antibody conjugates in widefield and GSDIM microscopy.
The following selected papers cite the use of and/or provide additional information about Chromeo 546:
- “Set of fluorochromophores in the wavelength range from 450 to 700 nm and suitable for labeling proteins and amino-modified DNA” by Wetzel et al (2003) Journal of Chromatography B 793:83-92.
- “A new Fluorescence Resonance Energy Transfer Pair and its Application to Oligonucleotide Labeling and Resonance Energy Transfer Hybridization Studies” by Gruber et al (2005) J. of Fluorescence 15:207-214.
- “Probing the electromagnetic field of a 15-nanometre hotspot by single molecule imaging” by Canget al (2011) Nature 469:385-388.
The Chromeo 546 Antibody Labeling Kit has been cited in the following publications:
- “High Mobility Group Protein-1 Inhibits Phagocytosis of Apoptotic neutrophils through Binding to Phosphatidylserine” by Liu et al (2008) J. of Immunology 181:4240-4246.
Contents & Storage
Chromeo™ 546 NHS-Ester, Chromeo™ 546 Carboxylic Acid and Chromeo™ 546 Biotin 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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