ATTO dyes (ATTO-TEC) are a family of synthetic fluorophores spanning the UV to near-infrared spectrum, characterized by high molar extinction coefficients, high fluorescence quantum yield, and exceptional thermal and photostability. Available ATTO formats for anti-rabbit reagents include ATTO 390, 425, 488, 550, 565, 594, 647N, 680, and 700. The emission spectra match the principal output wavelengths of most common fluorescence instrumentation.
Applications
ATTO-conjugated anti-rabbit reagents are validated for Western blotting, immunofluorescence microscopy, immunohistochemistry, immunocytochemistry, flow cytometry, immunoprecipitation, and fluorescence-based plate assays (FLISA). ATTO dyes are particularly suited to STED super-resolution microscopy and FRET, owing to their high photostability and strong quantum yield. Working dilutions vary by conjugate; representative values for an ATTO 425 anti-rabbit reagent are >1:10,000 for Western blot, >1:5,000 for immunofluorescence, and >1:20,000 for FLISA.
Specificity
For secondary antibody formats, specificity is defined by the immunoglobulin class targeted—typically rabbit IgG heavy and light chains. Reagents are prepared by immunoaffinity chromatography on immobilized rabbit IgG followed by solid-phase adsorption to remove unwanted reactivities; immunoelectrophoresis yields a single precipitin arc against goat serum, rabbit IgG, and rabbit serum, with no reaction against bovine, chicken, goat, guinea pig, hamster, horse, human, mouse, rat, or sheep serum proteins. The dye-to-protein ratio is typically ≥2, and unconjugated dye is ≤5% of total fluorescence. For directly conjugated rabbit primary antibodies, specificity is determined by the clone or antiserum rather than by the ATTO label.
