TrueBlot® Immunoprecipitation and Western Blot Kit for 6X HIS Epitope Tag

TrueBlot® Immunoprecipitation and Western Blot Kit for 6X HIS Epitope Tag allows for the immunoprecipitation and detection of 6X HIS-tagged recombinant proteins present in cell lysates provided by the user. After protein separation by SDS-PAGE and transfer, the membrane is probed with monoclonal Anti-6X HIS. Detection of the membrane bound antibody-antigen complex is achieved by the addition of Mouse IgG TrueBlot® ULTRA HRP. The TrueBlot® Immunoprecipitation and Western Blot Kit for 6X HIS Epitope Tag contains the critical supporting reagents, buffers, and substrates for immunoprecipitation and Western blotting of samples containing a 6X HIS Epitope Tag using TrueBlot monoclonal secondary antibody in conjunction with Rockland's Anti-6X HIS (MOUSE) Monoclonal Antibody. Mouse IgG TrueBlot® ULTRA is the unique horseradish peroxidase conjugated Anti-Mouse IgG monoclonal secondary antibody which enables detection of immunoblotted target protein bands, without hindrance from interfering immunoglobulin heavy and light chains from your IP antibody. Use it in place of your conventional HRP Anti-Mouse IgG immunoblotting secondary antibody. It is easy to generate publication-quality IP/WB data with Mouse IgG TrueBlot® ULTRA. Mouse IgG TrueBlot ULTRA is ideal for use in protocols involving immunoblotting of immunoprecipitated proteins. TrueBlot preferentially detects the non-reduced form of mouse IgG over the reduced, SDS-denatured form of IgG. When the immunoprecipitate is fully reduced immediately prior to SDS-gel electrophoresis, reactivity of Mouse IgG TrueBlot ULTRA 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/immunoblotting applications. Applications include studies examining post-translational modification (e.g., phosphorylation or acetylation) or protein-protein interactions. Mouse IgG TrueBlot® ULTRA 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). Mouse IgG TrueBlot® ULTRA is an HRP-conjugated monoclonal secondary antibody reacting with mouse IgGs for optimal signal detection in immunoprecipitation/immunoblotting experiments. Note that there are three key procedural considerations: 1. Protein A or G beads may be used with the mouse, goat and sheep TrueBlot secondaries, but not with the rabbit TrueBlot secondary. Use of protein A or G beads with the rabbit TrueBlot will result in contaminating bands. 2. Immunoprecipitate should be completely reduced. 3. BLOTTO/Milk should be used as the blocking protein for the immunoblot. TrueBlot® IP and WB Kit for 6X HIS Epitope Tag Components: 1. Mouse IgG TrueBlot ULTRA: 50 µL 18-8817-31; 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. Anti-Mouse Ig IP Beads: 2.5 mL Binds 0.4 mg Ig/mL beads; 8. Anti-6X HIS (MOUSE) Monoclonal Antibody: 100 µg; 9. Western Blot Incubation Tray. Special Notes: Upon initial use of the IP beads, we recommend that the vial be inverted several times to get the beads into suspension. We recommend using a large bore pipet to pipet up the liquid for use. For storage of the opened vial of beads, we recommend that the vial cap be sealed with parafilm to help prevent evaporation of the buffer. 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.