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TrueBlot
Immunoprecipitation (IP) and Western Blot (WB) protocols provide highly specific results but often suffer from poor or non-specific binding, high background, and heavy/light chain interference. Rockland’s TrueBlot® line of products consists of both TrueBlot® IP beads and TrueBlot® monoclonal secondary antibodies, which combat these common experimental issues by providing increased specificity, low background, and enhanced accuracy to provide publication-quality IP Western Blots without interference from contaminating IgG.
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How TrueBlot® Secondaries Work
When proteins are isolated using IP, a capture antibody binds to the target protein (Step 1), Protein A- or Protein G-coated magnetic beads are added to immobilize the antibody-target protein complex (Step 2) and the undesired material is washed away (Step 3). The final steps in a conventional IP typically include denaturation of the proteins using heat and a reducing/denaturing agent (Step 4).
When the eluted IP sample is separated via SDS-PAGE (Step 5), probed with the same primary antibody used in IP, and detected with conventional secondary antibodies in Western blot, both the heavy and light chain of the IP capture antibody can be detected (Step 5; right panel), which can obscure your protein of interest, specifically if it is similar in size to the heavy or light chain.
TrueBlot® secondary antibodies only recognize primary antibodies in their native (non-reduced) state, eliminating interference from heavy and light chain bands (Step 6; left panel) to provide accurate, publication-quality Western blots of immunoprecipitated samples.
When to Use TrueBlot® Products
The TrueBlot® product line is ideal for studying protein-protein interactions, post-translational modifications (phosphorylation and acetylation), poorly expressed proteins, and immunoprecipitated proteins with molecular weights similar to the heavy and light chain.
TrueBlot secondary antibodies preferentially detect the native, non-reduced form of IgG over the reduced form of IgG, making them ideal for Western blots of immunoprecipitated proteins. These unique detection properties reduce interference by the heavy chain (~55 kDa) and light chain (~23 kDa) of the immunoprecipitating antibody enabling unhindered detection of blotted target bands. Pair TrueBlot® antibodies with the exceptional purification properties provided by TrueBlot® IP beads to eliminate the common issues of IP-Western Blot.
TrueBlot® IP beads and TrueBlot® monoclonal secondary antibodies are available in a variety of host species and can be purchased individually, in sets with the corresponding agarose Ig IP beads, or as Western blot kits, allowing researchers the ability to choose a TrueBlot® product to fit all their research needs.