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Secondary Antibody Conjugates
anti-rabbit & anti-mouse secondaries for enhanced HRP and fluorescent detection
Active Motif's secondary antibody conjugates offer an improved method for the detection of primary antibodies. Having developed many quality primary antibodies, we found that the quality of secondary antibodies was also key to producing superior results. We therefore offer these secondary antibodies, conjugated to a number of top-quality molecules, including HRP and the Chromeo™ line of fluorescent dyes, giving you a variety of choices for detecting primary antibodies bound to their targets. To conjugate the Chromeo Dyes to primary antibodies and other proteins, we offer our complete, optimized antibody/protein labeling kits.
Figure 1: 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 (Cat. No. 39117) and Chromeo 642 Goat anti-rabbit IgG (Cat. No. 15044, below), while tubulin was visualized using alpha Tubulin mouse mAb (Clone 5-B-1-2) and Chromeo 488 Goat anti-mouse IgG (Cat. No. 15031, below). The nuclei were counter-stained with DAPI, then the three separate images were merged.
Active Motif's ATTO dye conjugates have been maximally cross-adsorbed against IgG's of a variety of species to eliminate background caused by non-specific binding. The fluorescent ATTO (STED) secondary antibody conjugates and the fluorescent Chromeo™ 488, Chromeo™ 505 and Chromeo™ 494 secondary antibody conjugates have been certified by Leica Microsystems for STimulated Emission Depletion (STED) microscopy, as they exhibited superior staining when compared to other fluorescent antibody conjugates. The ATTO 532 (GSD) and the Rhodamine 6G (GSD) secondary antibody conjugates are recommended by Leica Microsystems for high resolution GSDIM microscopy using the novel SR GSD microscope.
Chromeo™ Dyes are bright fluorescent labels that replace Alexa Fluor*, DyLight* or Cy*-dyes. They are compatible with most excitation sources including diode lasers, LEDs, tungsten lamps and xenon arc lamps. The dyes have been conjugated to high-quality secondary antibodies by an optimized conjugation method, including subsequent purification from interfering substances.
Chromeo™ Dye Secondary Antibody Conjugate advantages
- High intensity
- Specificity under various fixation conditions
- Photostability – the combination with MAX Stain™ reagents provides optimal fluorescent stability for multiple exposures and increased exposure time
- Low background
Fluorescent Chromeo Dyes Secondary Antibody Conjugates enable sensitive and specific detection in fluorescence microscopy, high content screening, ELISA, FRET applications or flow cytometry. To ensure that you get the best results possible, we highly recommend that you use our secondary antibody conjugates together with our MAX Stain™ Immunofluorescence Tools, as those components have been formulated to optimize the performance of the Chromeo™ Dyes.
To receive more detailed information and application data about individual Chromeo dyes, simply click on the name of the Chromeo dye of your choice in the table below. To see a larger spectra image, click on the spectrum of your choice.
| Dye | Absorption | Emission | Spectra | ε L/(mol-cm) | Stokes shift |
|---|---|---|---|---|---|
| Chromeo™ 488 | 498 nm | 524 nm |  |
73,000 | 26 nm |
| Chromeo™ 494 | 489 nm | 624 nm |  |
55,000 | 135 nm |
| Chromeo™ 505 | 514 nm | 530 nm |  |
70,000 | 16 nm |
| Chromeo™ 546 | 550 nm | 567 nm |  |
98,800 | 17 nm |
| Chromeo™ 642 | 647 nm | 666 nm |  |
180,000 | 19 nm |
| ATTO 594 | 601 nm | 627 nm |  |
120,000 | 26 nm |
| ATTO 647N (STED) | 644 nm | 669 nm |  |
150,000 | 25 nm |
| ATTO 655 (STED) | 663 nm | 684 nm |  |
125,000 | 21 nm |
| ATTO 532 (GSD) | 534 nm | 560 nm |  |
115,000 | 26 nm |
| Rhodamine 6G (GSD) | 508 nm | 558 nm |  |
116,000 | 50 nm |
| Table 1: Properties of Active Motif's Fluorescent Antibody Conjugates. | |||||
Next to the spectral properties of the dye and the quality of the secondary antibody, the quality of a fluorescent conjugate is influenced by the dye-to protein ratio, the conjugation method and its purity. All Active Motif Secondary Antibody Conjugates have been prepared by an optimized conjugation protocol making the fluorescent secondaries brighter and lowering the fluorescent background. Active Motif antibody conjugates have been tested in various applications including flow cytometry and fluorescent microscopy where they have shown to work with high efficiency and specificity under multiple fixation conditions.
Active Motif Fluorescent Secondary advantages
- Unrivaled fluorescent intensity
- Low background
- Limited photobleaching
- High Specificity
- Flexibility
Figure 1: Chromeo 642 staining in HeLa cells.
HeLa cells were stained with alpha Tubulin mouse mAb (Clone 5-B-1-2) and Chromeo 642 Goat anti-mouse IgG (Catalog No. 15034). The nuclei have been counterstained with DAPI.
Figure 2: Chromeo 488 antibody conjugates in 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 (left). Vimentin was stained with a primary polyclonal rabbit antibody and with Chromeo 488 Goat anti-rabbit IgG (Catalog No. 15041) secondary antibody (right). These STED images are courtesy of Leica Microsystems, Germany.
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 Chromeo 546 Goat anti-mouse IgG (Catalog No. 15033).
Contents & Storage
HRP conjugated secondaries are supplied as 400 µg of antibody at a concentration of 0.8 µg/µl, in 30% glycerol. Store the antibody at -20°C. These products are guaranteed for 6 months from the date of arrival.
The Chromeo™ conjugates are supplied as 1 mg of antibody at a concentration of 2 mg/ml. The ATTO and the Rhodamine 6G conjugates are supplied in 200 or 35 µl aliquots in PBS. For short-term storage, the conjugated antibody should be stored at 4°C protected from light. For longer-term storage, aliquot the antibody and store at -20°C. Avoid subjecting the antibody to repeated freeze-thaw cycles. These products are guaranteed for 6 months from the date of arrival.
To ensure photostability of Chromeo 488 under all experimental conditions, one aliquot of MAXfluor™ Mounting Medium for use in fluorescence microscopy is supplied with Chromeo 488 conjugates. This will provide optimal fluorescence stability and anti-fading during long-term storage of your mounted slides. MAXfluor Mounting medium should be stored at 4°C.
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