Mouse Pure-Blot anti-Rabbit IgG (H+L) Secondary Antibody (eB182) [DyLight 800]

This secondary antibody has been tested in dot blot, western blot, and immunoprecipitation and may also be used for detection in immunoassays that do not employ immunoprecipitation. This secondary antibody is provided as a lyophilized powder. To conserve reagent, we recommend incubating the blots from minigels in sealed bags (removing as much air as possible) with minimal volume (2-3 mLs). If used conservatively at 2.5mLs/blot will yield enough reagent for 200 blots. Note that there are three key procedural considerations: 1. Protein A or G should not be used for the immunoprecipitation. Use of protein A or G beads with the rabbit Pure-Blot will result in contaminating bands. For immunoprecipitation, Anti-rat IgG beads, or Anti-rabbit IgG beads should be used for rat or rabbit immunoprecipitating antibodies, respectively. 2. Immunoprecipitate should be completely reduced. 3. Bovine Serum Albumin, or blocking buffer for Fluorescent Western Blotting, at low concentrations, should be used as the blocking protein for the immunoblot. DO NOT USE BLOTTO or MILK. All recommended dilutions for listed applications are intended as an initial recommendation, specific conditions for each protein and antibody combination should be specifically optimized by the end user. 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. DyLight(TM) 800 and DyLight(TM) 680 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. DyLight(TM) 800 conjugates are optimized for the Odyssey(R) Infrared Imaging System developed by LI-COR. DyLight(TM) 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 DyLight(TM) 800 conjugates are used in conjunction with DyLight(TM) 680 conjugates. DyLight(TM) 800 and DyLight(TM) 680 conjugates provide an ultra-sensitive and convenient alternative to standard chemiluminescent protein detection methods, as well as a valuable tool for multicolor imaging.