Mouse IgG (H&L) Antibody IRDye800CW(R) Conjugated

Fluorescence technology is widely used to detect proteins. However, many common visible fluorophores often result in considerable background fluorescence in the visible range. Visible fluorophores are rarely used for membrane-based protein detection because of this high background. IRDye(R) 800 and IRDye(R) 700DX antibody and reagent conjugates are specifically designed for protein detection methods that use longer-wavelength, near-infrared (IR) fluorophores to visualize proteins in western blotting and other applications. Very low background fluorescence in the IR range provides for a much higher signal-to-noise ratio than visible fluorophores. Detection levels in the picogram range on Western blots rival the sensitivity of chemiluminescence on film. IRDye(R) 800 conjugates are optimized for the Odyssey(R) Infrared Imaging System developed by LI-COR. IRDye(R) 800 conjugates are also suitable for immunofluorescence microscopy using commercially available excitation/emission filters in the 780nm/820nm range. Dual simultaneous labeling in western blots or microscopy is achieved when IRDye(R) 800 conjugates are used in conjunction with IRDye(R) 700DX or DyLight(TM)680 conjugates. IRDye(R) 800 and IRDye(R) 700DX conjugates provide an ultra-sensitive and convenient alternative to standard chemiluminescent protein detection methods, as well as a valuable tool for multicolor imaging. IRDye(TM)800CW conjugates are specifically designed for LI-COR's In-Cell Western assay for the Odyssey(R) Infrared Imaging System. IRDye(TM)800CW conjugates can be used for western blotting applications, but the fluorochrome/protein ratio is not optimal for this application so detection sensitivity may be reduced. The In-Cell Western assay is a faster method for analyzing signal transduction pathways. In less time than a gel-based Western, you can quantify proteins in a 96- or 384-well microplate. In-Cell Westerns use infrared dye-labeled secondary antibodies to directly label proteins in fixed cultured cells, and quantify total fluorescence from each well. Time consuming and error-prone steps such as lysate preparation, gel loading and electrophoresis, and membrane transfer are eliminated with In-Cell Westerns. Simultaneous, two-target detection enables quantitative and accurate measurement of abundance or phosphorylation of one target by normalization against another target. Near-infrared probes yield high sensitivity for measuring small changes in protein amount or modification. Direct detection of proteins in their cellular context eliminates artifacts caused by cell lysis. The fast, microplate-based assay eliminates lysates, gels, and membranes required for conventional Western blots. The In-Cell Western assay is a moderate throughput method, ideal for screening cell treatments or drug candidates for their effects on target proteins. Recommended plates for In-Cell Westerns are Nunc-Nalgene (p/n 167008) for 96-well format and Falcon (BD Biosciences p/n 353961) for 384-well format. Primary and secondary antibodies should be validated by Odyssey western blotting before performing the In-Cell Western assay to confirm specificity and performance of the reagent in the infrared system.