Secondary antibodies conjugated with DyLight Fluors and highly purified for superior multiplex fluorescent cell imaging.

Thermo Scientific DyLight Labeled Highly Cross-Adsorbed Secondary Antibodies are fluorescent anti-mouse and anti-rabbit IgG antibodies that are specially purified to minimize cross-reactivity with immunoglobulins of other species.

These DyLight Fluor Conjugated Antibodies are especially useful for multiplex experiments in which primary antibodies from two or more host species are used simultaneously. They are designed to reduce nonspecific background staining and offer high specificity, low background and increased assay sensitivity. With these conjugates, incubation times are reduced and there is good lot-to-lot consistency.

The affinity-purified goat anti-mouse IgG (H+L) are solid-phase adsorbed to minimize cross-reaction with human, bovine, horse, rabbit and swine serum proteins. The affinity-purified goat anti-rabbit IgG (H+L) are solid-phase adsorbed to minimize cross-reaction with human, mouse and rat serum proteins.

DyLight Conjugate Highlights:

• Anti-mouse and anti-rabbit antibodies – polyclonal goat antibodies provide species-specific detection of the entire target IgG class (heavy and light chains, H+L)
• Higly cross-adsorbed – affinity-purified goat anti-mouse and anti-rabbit IgG (H+L) antibodies were solid-phase adsorbed to minimize cross-reaction with human, bovine, horse, rabbit and swine serum proteins
• Optimized for conjugate performance – reagents, labeling (dye:protein ratio) and purification methods are optimized to provide the highest possible binding function and overall fluorescence intensity
• Consistent and stable – all conjugates are stable for 1 year at 4°C, providing consistent performance for the duration of use
• Application-ready – excellent for flow cytometry, fluorescence microscopy, ELISA, protein arrays and high content analysis (HCA)

DyLight Fluorophore Highlights:

• Bright fluorescence – intense emission provides superior sensitivity and requires less conjugate
• Narrow emission spectra – negligible bleed-through between fluorophore channels enables multi-color detection
• Excellent photostability – exceptional resistance to photobleaching enables fluorescence imaging under the most demanding conditions (e.g., structured illumination, 4Pi microscopy)
• Buffer stability – conjugated fluorophores are completely stable at pH 4-9
• Instrument compatibility – excitation and emission spectra correspond with filter sets and laser settings of all popular fluorescence instrumentation

Emission spectra of Thermo Scientific DyLight Fluors 350-800. Line colors represent the approximate visible colors of the wavelength maxima. Learn more... DyLight Dye spectral properties (opens new window)

Details and Performance Data:

Each highly cross-adsorbed antibody conjugated to a DyLight Dye was tested for cross-reactivity in plate-based IgG binding assays. They did not exhibit any cross-reaction to human, bovine, horse, rabbit, swine or rat IgGs. The degree of cross-reactivity was determined by ELISA and was typically less than 1% of the desired signal. Our goat anti-mouse conjugates showed low levels of crossreactivity with rat IgG; however, similar levels of cross-reactivity were observed in other commercially available conjugates.

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Comparison of Thermo Scientific DyLight Fluors conjugated to highly cross-adsorbed secondary antibodies with equivalent Alexa Fluor* Dye-Conjugated Antibodies. Intermediate filaments stained in A549 cells using a rabbit polyclonal anti-cytokeratin 18 antibody (0.103µg/mL) and detected with GAR IgG highly cross-adsorbed labeled secondary antibodies (2µg/mL) labeled with A. DyLight Fluor 488 (Product # 35553), D. Alexa Fluor 488 (green), B. DyLight Fluor 549 (Product # 35558), E. Alexa Fluor 555 (orange), C. DyLight Fluor 649 (Product # 35566) and F. Alexa Fluor 647 (yellow). Nuclei are counterstained with Hoechst (Product #33342, blue).

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Multiplex immunostaining of mouse anti-lamin A and rabbit anti-cytokeratin 18 primary antibodies detected with Thermo Scientific DyLight Fluors conjugated to secondary antibodies in A549 cells. Co-detection was performed with A. DyLight 405-GAM and DyLight 633-GAR highly cross-adsorbed, B. DyLight 488-GAM highly cross-adsorbed and DyLight 405-GAR highly cross-adsorbed or C. DyLight 549-GAM highly cross-adsorbed and DyLight 680-GAR highly cross-adsorbed. Acquired images show uniform intermediate filaments staining of cytokeratin 18 pseudo-colored in red (DyLight 633), blue (DyLight 405) or white (DyLight 680) located exclusively in the cytoplasm and a uniform nuclear membrane staining of lamin A, pseudo-colored in blue (DyLight 405), green (DyLight 488) or orange (DyLight 549).

Co-detection of Mouse anti-Muc5AC and rabbit anti-E-cadherin using Thermo Scientific DyLight Fluor 488-GAM and DyLight Fluor 549-GAR highly cross-adsorbed IgG (H+L). Normal bronchial epithelial cells differentiated at the air-liquid interface were probed simultaneously with mouse anti-Mu5AC and rabbit anti-E-cadherin and then were visualized with DyLight Fluor 488-GAM and DyLight Fluor 549-GAR, respectively. Images show intense staining of Muc5AC expressing cells (Green) and the network of membrane bound E-cadherin (Red).

Co-detection of mouse anti-a-tubulin and rabbit anti-lamin B1 using Thermo Scientific DyLight Fluor 594-GAM and DyLight Fluor 488-GAR Highly Cross-Adsorbed IgG (H+L). HeLa cells were probed simultaneously with mouse a-tubulin and rabbit anti-lamin B1 primary antibodies at 10µg/mL and 0.4µg/mL, respectively, and then were visualized after incubation with a mixture of DyLight Fluor 594-GAM highly cross-adsorbed and DyLight Fluor 488-GAR highly cross-adsorbed both at 4µg/mL. Acquired images show bright microtubules spindles (Red) and lamin B1 (Green) in HeLa cells.

Related Products and Resources:

• Complete DyLight Fluor Product Guide
• Fluorescent Labeling Reagents and Kits
• DyLight Fluor Secondary Antibody Conjugates (not cross-adsorbed)
• DyLight Fluor Streptavidin and NeutrAvidin Conjugates

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