Rockland’s TrueBlot® product line is designed to solve common experimental problems when performing immunoprecipitation/co-immunoprecipitation experiments and Western blots of immunoprecipitated samples (IP–Western blot). The product line consists of TrueBlot® monoclonal secondary antibodies and TrueBlot® IP beads.

TrueBlot® antibodies are specific to the whole IgG molecule and will not bind heavy or light chains. This is useful for binding target proteins with an expected MW near 25 kDa (light chains) and 50 kDa (heavy chains).

The sample must be fully reduced to eliminate cross-reactivity with heavy and light chains. Any reactivity to heavy and light chains could be due to incomplete reduction of your sample. Please be sure to optimize reducing conditions for your sample type.

Samples can be fully reduced by heating at 90-100°C for 10 minutes in sample buffer containing reducing agent (for example, DTT to a final concentration of 50 mM or add β-Mercaptoethanol to a final concentration of 2% (v/v)).

TrueBlot® secondary antibodies are reactive with the IgG of their respective species. For example, if using a primary antibody raised in mouse (mouse host), use TrueBlot® anti-mouse Ig secondary antibodies for detection.

TrueBlot® secondary antibodies have not been shown to detect IgM.

Samples that have many other proteins present (such as lysates) may require preclearing to prevent interference in later IP and WB procedures. Recombinant protein samples require pre-clearing more often than serum samples.

The immunoprecipitation procedure can be repeated several times to yield a more concentrated protein solution.

The optimal lysis buffer will depend on your sample type. See protocol for buffer recommendations. Generally, 1X RIPA is used to lyse samples.

Be sure to include a positive control for the primary antibody in your experiment and check that the sample is fully reduced.

The beads are roughly 0.5 μm in diameter.

Using Protein A or Protein G beads with rabbit species results in contaminated bands. For best results when using rabbit species, use Rockland’s TrueBlot® Rabbit Ig IP beads, which are available in either a magnetic or agarose format. Please see individual TrueBlot® Rabbit product pages for additional details.