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Chromeo™ 505

effective green fluorescent dye for bioanalysis

Chromeo™ 505 is a bright, green fluorescent label that replaces Alexa Fluor 500*, DyLight 505* and other dyes excitable at a wavelength of 488 nm, such as fluorescein and FITC. The dye, which is compatible with filter sets designed for fluorescein, is a good choice to replace traditional green-emitting fluorophores and their conjugates.

Why use Chromeo™ 505?

  • 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 505 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. In addition Leica Microsystems recommends Chromeo 505 for use in STED microscopy because it meets the specifications required for STED microscopy, where it provides brighter, higher resolution pictures than other 488 nm-excitable dyes. For your convenience, Chromeo 505 is offered as optimized Fluorescent Secondary Antibody Conjugates for enhanced detection.

To ensure the highest quality in your immunofluorescent staining, we recommend the use of Chromeo 505 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™ 505 Carboxylic Acid 1 mg 15610 $115 Buy Now
5 x 1 mg 16610 $445 Buy Now
Chromeo™ 505 NHS-Ester 1 mg 15611 $230 Buy Now
5 x 1 mg 16611 $865 Buy Now
 
Chromeo 505 Fluorescent Dye Spectra
 
Figure 1: Absorption and emission spectra of Chromeo™ 505.

 

Dye Absorption Emission ε L/(mol-cm) Stokes shift
Chromeo™ 505 505 nm 526 nm 70,000 21 nm
Table 1: Properties of Chromeo™ 505.
 

Chromeo™ 505 is a bright, green fluorescent dye that can be excited at 488 nm. The dye is very stable over a broad pH range and in combination with the MAX Stain™ reagents it shows high fluorescence intensity and photostability. Chromeo 505 and Chromeo 505 Fluorescent Secondary Antibody Conjugates are a good choice to replace traditional green-emitting fluorophores.

HeLa cells stained by alpha-Tubulin mAb and Chromeo 505 Fluorescent Secondary Antibody
Figure 1: Chromeo 505 staining in HeLa cells.

HeLa cells were stained with alpha Tubulin mouse mAb (Clone 5-B-1-2) and Chromeo 505 Goat anti-mouse IgG (Catalog No. 15030).

Even if the emission spectrum of Chromeo 505 is shifted towards longer wavelength by some nm, the dye and its conjugates can still be used with filters designed for fluorescein. This makes Chromeo 505 a good candidate for multi-color staining experiments with other fluorescent dyes in the yellow or red fluorescent range. Figure 2 shows a co-staining experiment using Chromeo 505 in combination with Chromeo 642 and DAPI as a nuclear stain. Figure 3 shows Chromeo 505 staining in combination with the Hoechst nuclear stain.

Concurrent multi-color staining of Phospho H2AX and tubulin using Chromeo 505 and Chromeo 642 secondary antibodies
Figure 2: Concurrent staining using Chromeo 505 and Chromeo 642 fluorescent secondaries in untreated and etoposide-treated HeLa cells.

HeLa cells were either left untreated or treated with 100 µM etoposide for 6 hours prior to fixation with methanol. The histone-variant H2AX was stained with Histone H2AX phospho Ser139 rabbit pAb (Catalog No. 39117) and Chromeo 642 Goat anti-rabbit IgG (Catalog No. 15044), while tubulin was visualized using alpha Tubulin mouse mAb (Clone 5-B-1-2) and Chromeo 505 Goat anti-mouse IgG (Catalog No. 15030). The nuclei were counter-stained with DAPI and the three separate images were merged.

Staining of Oct-4 using Chromeo 505 secondary antibody
Figure 3: Oct-4 staining using Chromeo 505 fluorescent secondary in human IMR-90 cells.

The transcriptional activator Oct-4 was stained with Oct-4 rabbit polyclonal antibody (Catalog No. 39811) and Chromeo 505 Goat anti-rabbit IgG (Catalog No. 15040) (A). The nuclei were counter-stained with Hoechst (B).

In contrast to fluorescein, Chromeo 505 shows high photostability in fluorescence microscopy, independently of the reagents used for sample preparation. This was demonstrated by staining tubulin in HeLa cells with reagents containing either PBS or MAX Stain™ reagents. The cells were excited over a period of 90 seconds: images were taken with an exposure time of 250 milliseconds with an interval of 735 milliseconds between the images. Figure 4, presenting the first picture taken in each series and picture number 100 of each time course, illustrates the high level of photostability of the 488 nm excitable dye in either buffer type.

Time course demonstrating photostability of Chromeo 505 in PBS and MAX Stain reagents
Figure 4: Time course demonstrating the photostability of Chromeo 505 in PBS and MAX Stain reagents.

HeLa cells were stained with alpha Tubulin mouse mAb (Clone 5-B-1-2) and Chromeo 505 Goat anti-mouse IgG (Catalog No. 15030). The two samples were prepared using either a BSA-based buffer system or MAX Stain Immunofluorescence reagents. The first and 100 images in each time course are shown.

Chromeo 505 has been shown to work in several different types of fluorescence microscopy. In addition to classical widefield applications, Chromeo 505 and its Fluorescent Secondary Antibody Conjugates have been tested in confocal scanning microscopy and high resolution microscopy. For STED (STimulated Emission Depletion) microscopy using the Leica TCS STED CW microscope, Active Motif’s fluorescent Chromeo 505 dye and conjugated secondary antibodies have been certified by Leica Microsystems. The fluorescent properties, especially the high photostability of Chromeo 505 dye and secondaries meet the specifications required to perform STED microscopy, as shown below by staining of nuclear targets (Figures 5 and 6).

Nuclear pore protein-1 (NUP-1) stained with a primary monoclonal mouse antibody and Chromeo 505
Figure 5: Chromeo 505 antibody conjugates in STED microscopy.

Nuclear pore proteins stained with a primary monoclonal mouse antibody and Chromeo 505 Goat anti-mouse IgG (Catalog No. 15030) secondary antibody. The images on the left were prepared using a confocal microscope, while those on the right were produced using a STED microscope. The bottom images are close ups of the top images. All images are courtesy of Leica Microsystems, Germany.

Use of Active Motif's primary alpha Tubulin and H3K4me3 antibodies and Chromeo 505 fluorescent secondary antibody in STED microscopy
Figure 6: Active Motif's primary and fluorescent secondary antibodies in STED microscopy.

HeLa cells were stained with alpha Tubulin mouse monoclonal antibody (Clone 5-B-1-2) (Catalog No. 39527), a biotinylated secondary goat anti-mouse antibody and BD Horizon V500-streptavidin conjugate. Histone H3 was stained with Histone H3 trimethyl Lys4 rabbit polyclonal antibody (Catalog No. 39159) and Chromeo 505 Goat anti-rabbit IgG (Catalog No. 15040) secondary antibody. The STED image is courtesy of Leica Microsystems, Germany.

Chromeo 505 has been shown to work in a second super-resolution microscopy technology. For GSDIM (Ground State Depletion with Individual Molecule return) microscopy using the Leica GSD SR microscope, Active Motif’s fluorescent Chromeo 505 dye and conjugated secondary antibodies have been tested and recommended by Leica Microsystems. The fluorescent properties of Chromeo 505 meet the specifications required to perform GSDIM microscopy, using the 488 nm/300 mW laser for excitation (Figure 7).

Comparison of conventional widefield microscopy and GSDIM microscopy using Chromeo 505 Goat anti-mouse IgG secondary antibody
Figure 7: Chromeo 505 antibody conjugates in widefield and GSDIM microscopy.

Tubulin was stained with a primary monoclonal mouse antibody and with Chromeo 505 Goat anti-mouse IgG (Catalog No. 15030) secondary antibody. The widefield (left) and GSDIM (right) images are courtesy of Leica Microsystems, Germany.

Chromeo 505 conjugated Fluorescent Secondary Antibodies have been cited in the following publication:

Contents & Storage

Chromeo™ 505 NHS-Ester and Chromeo™ 505 Carboxylic Acid are supplied lyophilized and should be stored at the recommended temperature in the dark to ensure photostability.

All products are guaranteed 6 months when stored properly.

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