TrueBlot monoclonal secondary antibodies for IP-WB

The TrueBlot® monoclonal secondary antibodies from Rockland Immunochemicals accurately detect immunoblotted native target protein bands, eliminating interference from denatured IgG in immuno-precipitated (IP) samples to generate publication quality IP-Western blots. With over 1500 publications, these easy-to-use antibodies can be substituted for conventional workflow secondary antibodies to produce high quality images suitable for reproduction.

Benefits of using TrueBlot monoclonal secondary antibodies
• Accurate detection of IP samples
• Detect only native IgG
• Easy-to-use
• Available in HRP-conjugated forms targeting mouse, rabbit, goat or sheep IgG
• Available in fluorescent-conjugated forms targeting mouse or rabbit IgG
• Can be used in WB experiments without an IP step
• Ideal for studying post-translational protein modifications & protein-protein interactions

TrueBlot secondary antibodies in IP–Western blot experiments: How do TrueBlot antibodies work?

Western blots of immunoprecipitated samples can frequently result in the detection of unwanted background signals when the same antibody is used as the capture antibody in immunoprecipitation (IP) and the primary antibody in Western blot.

Following formation of the immune complex, incubation of the complex with IP beads and wash steps (Steps 1–3), the final steps in a conventional IP experiment typically employ a heating step in the presence of reducing/denaturing reagents (Step 4).

When the eluted IP sample is separated via SDS-PAGE, probed with the same primary antibody used in IP and detected with conventional secondary antibodies in WB, both the heavy and light chain of the IP capture antibody can be detected (Step 5; right panel), which can obscure the target protein of interest if it is similar in size to the heavy or light chain.

TrueBlot® secondary antibodies only recognise primary antibodies in their native (non-reduced) state, eliminating interference from heavy and light chain bands (Step 5; left panel) to provide accurate, publication-quality Western blots of immunoprecipitated samples.

Illustration of the detection of caspase 7 using HRP-conjugated Mouse TrueBlot® secondary antibody vs. a conventional HRP-conjugated anti-mouse Ig secondary antibody following a typical IP procedure.

Applications
• Immuno-precipitation
• Immuno-precipitation- Western Blot (IP-WB)
• Co-immunoprecipitation

Material available for download
TrueBlot Secondary Antibodies (HRP) protocol
Selecting TrueBlot products

TrueBlot antibodies - tutorial for IP/WB