Chromeo™ 546

Figure 1: Absorption and emission spectra 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.

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.

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.

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.

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).

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).

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 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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