Rabbit TrueBlot® Western Blot Kit

Description

Background

The Rabbit TrueBlot® Western Blot Kit contains the critical supporting reagents, buffers, and substrates for immunoprecipitation and Western blotting of samples using TrueBlot monoclonal secondary antibody in conjunction with your own primary IP antibody and primary (Rabbit IgG) Western blotting antibody (see application note for kit components). Rabbit IgG TrueBlot® is the unique horseradish peroxidase conjugated Anti-Rabbit IgG monoclonal secondary antibody which enables detection of immunoblotted target protein bands, without hindrance by interfering immunoglobulin heavy and light chains from your IP antibody. Use it in place of your conventional HRP Anti-Rabbit IgG immunoblotting secondary antibody. It is easy to generate publication-quality IP/WB data with Rabbit IgG TrueBlot®. 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/immunoblot applications. Applications include studies examining post-translational modification (e.g., phosphorylation or acetylation) or protein-protein interactions. Rabbit IgG TrueBlot may also be used for detection in immunoblotting assays that do not employ immunoprecipitation.

Application Note

Rabbit IgG TrueBlot® is provided as 1000X solution. 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 20 blots. Rabbit IgG TrueBlot® HRP-conjugated monoclonal secondary antibody reacting with goat IgGs for optimal signal detection in immunoprecipitation/immunoblotting experiments. 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. 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. BLOTTO/Milk should be used as the blocking protein for the immunoblot. A sample of Anti-Alpha-Tubulin (MOUSE) Monoclonal Antibody Cat #(200-301-880S), has been provided to be used as a loading control at the user’s discretion. Dilutions are to be optimized by the user. Rabbit TrueBlot Western Blot Kit Components: 1. Rabbit IgG TrueBlot: 50 μL. 18-8816-31; 2. TrueBlot Enhancer Solution: 25 mL; 3. TrueBlot Blocker: 10 g; 4. TrueBlot Assay Buffer: 30 mL, 20X; 5. TrueBlot Substrate A: 12.5 mL; 6. TrueBlot Substrate B: 12.5 mL; 7. Western Blot Incubation Tray. 8. Anti-Alpha-Tubulin (MOUSE) Monoclonal Antibody - 200-301-880S.

Purity/Specificity

Rabbit TrueBlot® Antibody Peroxidase Conjugate was prepared from tissue culture supernatant by Protein G affinity chromatography. Assay by Immunoelectrophoresis resulted in a single precipitin arc against Anti-Goat Serum. Reactivity is observed against native Rabbit IgG by both Western blot and ELISA.