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Chromeo™ 488

effective green fluorescent dye for bioanalysis

Chromeo™ 488 is a bright, green fluorescent label that can be used in place of Fluorescein, FITC, Alexa Fluor 488*, DyLight 488* and other dyes excitable at a wavelength of 488 nm. The dye, which is compatible with filter sets designed for fluorescein, exhibits superior luminescence properties and has been tested in a variety of applications.

Why use Chromeo™ 488?

  • Photostability – enables multiple exposures and increased exposure time when used in combination with MAX Stain™ reagents
  • Bright fluorescence – sensitive thresholds for detection
  • Low background
  • pH insensitive
  • Easy to use – no harsh chemicals required for conjugation
  • Narrow emission and absorption bands – enables multi-color experiments
  • STED microscopy

Reactive fluorescent Chromeo 488 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 488 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. To provide you with flexibility in labeling your antibodies and proteins, Chromeo 488 is offered in a variety of chemistries. For greater convenience, Chromeo 488 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 photostability of Chromeo 488 under all experimental conditions, one aliquot of MAXfluor™ Mounting Medium for use in fluorescence microscopy is supplied with the dye. MAXfluor provides optimal fluorescence stability and anti-fading during long-term storage of your mounted slides. In addition, we highly recommend the use of Chromeo 488 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™ 488 Alkyne 1 mg 15290 $240 Buy Now
5 x 1 mg 15291 $925 Buy Now
Chromeo™ 488 Carboxylic Acid 1 mg 15510 $115 Buy Now
5 x 1 mg 16510 $445 Buy Now
Chromeo™ 488 NHS-Ester 1 mg 15511 $230 Buy Now
5 x 1 mg 16511 $865 Buy Now
Chromeo™ 488 Streptavidin 1 mg 15513 $210 Buy Now
5 x 1 mg 16513 $825 Buy Now
Chromeo 488 Fluorescent Dye Spectra
 
Figure 1: Absorption and emission spectra of Chromeo™ 488.

 

Dye Absorption Emission ε L/(mol-cm) Stokes shift
Chromeo™ 488 488 nm 517 nm 73,000 29 nm
Table 1: Properties of Chromeo™ 488.
 

Chromeo™ 488 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 488 and Chromeo 488 Fluorescent Secondary Antibody Conjugates are the best choice to replace traditional green-emitting fluorophores and their conjugates.

HeLa cells stained by alpha-Tubulin mAb and Chromeo 488 Fluorescent Secondary Antibody
Figure 1: Chromeo 488 staining in HeLa cells.

HeLa cells were stained with alpha Tubulin mouse mAb (Clone 5-B-1-2) and Chromeo 488 Goat anti-mouse IgG (Catalog No. 15031). The nuclei have been counterstained with DAPI.

We compared Chromeo 488 antibody conjugate to other commercially available secondary antibody conjugates with the same fluorescent range, Alexa 488*, DyLight 488* and ATTO-488 conjugated secondary antibodies. HeLa cells were labeled with an antibody against alpha-tubulin followed by detection with the fluorescently labeled secondary antibodies. All samples were processed equally, with MAX Stain reagents being used for all steps of the immunostaining procedure.

In the first experiment, the exposure times required for each sample to reach equal fluorescence intensity were measured. Figure 2 demonstrates that Chromeo 488 conjugates are extremely bright, with only the Alexa 488 anti-mouse conjugate on the same level of performance.

Exposure times of 488-excitable antibodies to reach equal fluorescence intensity
Figure 2: Exposure times (in ms) of various 488-excitable dyes to reach equal fluorescence.
 

In a second set of experiments, MAX Stain reagents were used to study the photostability of different 488-excitable antibody-dye conjugates. Again, anti-tubulin staining was performed in HeLa cells. The cells were excited over a period of 100 seconds: images were acquired every 547 milliseconds. Figure 3 is a plot of the fluorescence intensity of various antibody-dye conjugates relative to the illumination time. The graph demonstrates that all of the tested secondary antibodies show a good resistance to photobleaching when used with the MAX Stain reagents.

Photobleaching analysis of 488-excitable dyes
Figure 3: Photobleaching analysis of various 488-excitable dyes in immunofluorescence experiments using MAX Stain Immunofluorescence Tools.
 

The fluorescent properties of Chromeo 488, such as its intensity and sharp excitation & emission bands, and its ability to be combined with MAX Stain™ Immunofluorescence Tools make Chromeo 488 and its Fluorescent Secondary Antibody Conjugates excellent tools for multi-color experiments in widefield microscopy. Figure 4 shows a combination of Chromeo 488 with Chromeo 642 and DAPI. Figure 5 shows a combination of Chromeo 488 with Cy3.

Multi-color staining of Phospho H2AX and tubulin using Chromeo 488 and Chromeo 642 secondary antibodies
Figure 4: Multi-color staining of Phospho H2AX and tubulin using Chromeo 488 and Chromeo 642 secondary antibodies 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 488 Goat anti-mouse IgG (Catalog No. 15031). The nuclei were counter-stained with DAPI and the three separate images were merged.

Confocal microscopy of Bruchpilot and Res using Chromeo 488 and Cy3
Figure 5: Structural studies of the nervous system in Drosophila larvae.

Bruchpilot was stained with Chromeo 488 (red), Res was stained with Cy3 (green) and imaged by confocal microscopy. Image courtesy of Stephan Sigrist and Werner Fouquet, Freie Universität Berlin, Germany.

Chromeo 488 is also an excellent candidate for detection in fluorescent plate-based assays. Active Motif has developed several assays based on the brightness and sharp fluorescent bands of Chromeo 488-conjugated secondary antibodies. As an example, Figure 6 shows a result obtained using Active Motif's Mitosis Determination Assay, which is a simple, fast and accurate, 2-color fluorescent assay to determine the percentage of cells undergoing mitosis within a specified population. In this assay, a Chromeo 488-conjugated Goat anti-rat IgG is used to detect the highly specific phosphorylated histone H3 (Ser 28) (Clone HTA28) antibody to quantify the phosphorylation of serine 28 on histone H3, which occurs during mitotic chromatin condensation.

Exposure times of 488-excitable antibodies to reach equal fluorescence intensity
Figure 6: Single cell analysis of untreated versus paclitaxel-treated cell populations quantified using the IsoCyte™ benchtop laser scanning cytometer (MSD Analytical Technologies).

Panel A shows that few cells are undergoing mitosis in the untreated cell population. Panel B shows the shift in cell population to the right of the dividing line that is detected by the 488 signal following paclitaxel treatment.

Chromeo 488 has been shown to work in several different types of fluorescence microscopy. In addition to classical widefield applications, Chromeo 488 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 488 dye and conjugated secondary antibodies have been certified by Leica Microsystems. The fluorescent properties of Chromeo 488 dye and secondaries meet the specifications required to perform STED microscopy, as shown below by staining of nuclear targets (Figure 7) and visualization of cell structure proteins (Figure 8) or neuromuscular junctions (Figure 9).

Confocal and STED microscopy images of cells co-stained with NUP-1 and Chromeo 488 Goat anti-mouse IgG
Figure 7: Chromeo 488 staining of NUP-1 in confocal scanning and STED microscopy.

Nuclear pore protein-1 (NUP-1) was stained with a primary monoclonal mouse antibody and with Chromeo 488 Goat anti-mouse IgG (Catalog No. 15031) secondary antibody. The image on the left was taken using a confocal microscope, while that on the right shows the visualization of the nuclear protein by STED microscopy. Images courtesy of Leica Microsystems, Germany.

Confocal and STED microscopy images of cells co-stained with microtubulin and Chromeo 488 Goat anti-rabbit IgG
Figure 8: Chromeo 488 staining of microtubulin in confocal scanning and STED microscopy.

Vimentin was stained with a primary polyclonal rabbit antibody and with Chromeo 488 Goat anti-rabbit IgG (Catalog No. 15041) secondary antibody. The image on the left was captured under a confocal microscope, while that on the right shows the visualization of the microtubulin network by STED microscopy. Images courtesy of Leica Microsystems, Germany.

Staining of Bruchpilot in Drosophila larvae with fluorescent Chromeo 488 dye using the Leica TCS STED CW system
Figure 9: Immunofluorescent staining of neuromuscular junctions in drosophila larvae.

Bruchpilot was stained with Chromeo 488 and imaged by STED microscopy using the TCS STED CW system. Image courtesy of Stephan Sigrist and Werner Fouquet, Freie Universität Berlin, Germany.

Chromeo 488 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 488 dye and conjugated secondary antibodies have been tested and recommended by Leica Microsystems. The fluorescent properties of Chromeo 488 meet the specifications required to perform GSDIM microscopy, using the 488 nm/300 mW laser for excitation (Figure 10).

Comparison of conventional widefield microscopy and GSDIM microscopy using Chromeo 488 Goat anti-mouse IgG secondary antibody
Figure 10: Chromeo 488 antibody conjugates in widefield and GSDIM microscopy.

Tubulin was stained with a primary monoclonal mouse antibody and with Chromeo 488 Goat anti-mouse IgG (Catalog No. 15031) 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 488:

The Chromeo 488 Antibody Labeling Kit has been cited in the following publications:

Chromeo 488 conjugated Fluorescent Secondary Antibodies have been cited in the following publications:

Contents & Storage

Chromeo™ 488 NHS-Ester and Chromeo™ 488 Carboxylic Acid are supplied lyophilized and should be stored at the recommended temperature in the dark. The Chromeo™ 488 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.

The 1 ml vial of MAXfluor™ Mounting Medium, which is included with all Chromeo 488 dyes and conjugates, should be stored at 4°C in the dark.

All products are guaranteed 6 months when stored properly.

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