Application Note: Fluorescent Rabbit TrueBlot® Antibody DyLight™ 800 has been tested in dot blot, western blot, and immunoprecipitation and may also be used for detection in immunoassays that do not employ immunoprecipitation. Fluorescent Rabbit TrueBlot® Antibody DyLight™ 800 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 TrueBlot 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 MB-070 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™ 800 and DyLight™ 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™ 800 conjugates are optimized for the Odyssey® Infrared Imaging System developed by LI-COR. DyLight™ 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™ 800 conjugates are used in conjunction with DyLight™ 680 conjugates. DyLight™ 800 and DyLight™ 680 conjugates provide an ultra-sensitive and convenient alternative to standard chemiluminescent protein detection methods, as well as a valuable tool for multicolor imaging.
Clone ID: eB182
Concentration Value: 1.0 mg/mL
FLISA Dilution: User Optimized
Flow Cytometry Dilution: 1:2,000 - 1:10,000
Immunohistochemistry Dilution: User Optimized
IF Microscopy Dilution: 1:500 - 1:2,500
Western Blot Dilution: 1:1000
General Disclaimer Note: This product is for research use only and is not intended for therapeutic or diagnostic applications. Please contact a technical service representative for more information. All products of animal origin manufactured by Rockland Immunochemicals are derived from starting materials of North American origin. Collection was performed in United States Department of Agriculture (USDA) inspected facilities and all materials have been inspected and certified to be free of disease and suitable for exportation. All properties listed are typical characteristics and are not specifications. All suggestions and data are offered in good faith but without guarantee as conditions and methods of use of our products are beyond our control. All claims must be made within 30 days following the date of delivery. The prospective user must determine the suitability of our materials before adopting them on a commercial scale. Suggested uses of our products are not recommendations to use our products in violation of any patent or as a license under any patent of Rockland Immunochemicals, Inc. If you require a commercial license to use this material and do not have one, then return this material, unopened to: Rockland Inc., P.O. BOX 5199, Limerick, Pennsylvania, USA.
Physical State: Lyophilized
Purity and Specificity: Fluorescent Rabbit TrueBlot® Antibody DyLight™ 800 Conjugate was prepared from tissue culture supernatant by Protein G affinity chromatography. Assay by Immunoelectrophoresis resulted in a single precipitin arc against Anti-Rabbit Serum. Reactivity is observed against native Rabbit IgG by both Western blot and ELISA.
Background: Rabbit IgG TrueBlot® is a unique DyLight™ 800 conjugated Anti-rabbit IgG monoclonal secondary antibody. Rabbit IgG TrueBlot® enables detection of immunoblotted target protein bands, without hindrance by interfering immunoprecipitating immunoglobulin heavy and light chains. It is easy to generate publication-quality IP/Fluorescent Western Blot data with Rabbit IgG TrueBlot®, simply substitute the conventional DL800 Anti-rabbit IgG blotting reagent with Fluorescent Rabbit TrueBlot® Antibody DyLight™ 800 and follow the prescribed protocol for sample preparation and immunoblotting. Ideal for Li Cor Odyssey imaging as well as other IR and near IR imaging systems. Rabbit IgG TrueBlot® is ideal for use in protocols involving immunoblotting of immunoprecipitated proteins. TrueBlot preferentially detects the non-reduced form of rabbit IgG over the reduced, SDS-denatured form of IgG. When the immunoprecipitate is fully reduced immediately prior to SDS-gel electrophoresis, reactivity of Rabbit IgG TrueBlot® with the 55 kDa heavy chains and the 23 kDa light chains of the immunoprecipitating antibody is minimized thereby eliminating interference by the heavy and light chains of the immunoprecipitating antibody in IP/Western blot applications. Applications include studies examining post-translational modification (e.g., phosphorylation or acetylation) or protein-protein interactions.
Low Endotoxin: No
Other: User Optimized
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