For optimal results, use a shaking platform during incubation steps. Using large blocking and wash buffer volumes minimizes nonspecific signal. Use sufficient volumes of wash buffer, blocking buffer, antibody solution and substrate working solution to cover blot and ensure that it never becomes dry. Avoid using milk as a blocking reagent when using avidin/biotin systems because milk contains variable amounts of endogenous biotin, which causes high background signal. Blocking buffer also affects system sensitivity. Empirical testing is essential to determine the appropriate blocking reagent for each Western blot system, as cross-reactivity of the blocking reagent with antibodies causes nonspecific signal. To optimize antibody concentrations, perform a systematic dot blot analysis. Tanon High-sig ECL Western Blotting Substrate requires more dilute antibody concentrations than those used with precipitating colorimetric HRP substrates. To determine the appropriate concentrations, perform a systematic dot blot analysis. If you are currently using a SuperSignal Chemiluminescent Substrate, switching to Tanon High-sig ECL Western Blotting Substrate requires increasing antigen and antibody concentrations. Use the same blotting conditions when switching from Amersham ECL Substrate or Pierce ECL Substrate to Tanon High-sig ECL Western Blotting Substrate. The special formulation of Tanon High-sig ECL Western Blotting Substrate makes it the ideal substitute for Amersham ECL Substrate or Pierce ECL Western Blotting Substrate without the need for additional optimization of assay conditions. Blots can be repeatedly exposed to X-ray film or Chemiluminescence imaging system to obtain optimal results or stripped of the immunodetection reagents and re-probed. Tanon High-sig ECL Western Blotting Substrate enables the detection of picogram amounts of antigen and allows for easy detection of HRP using photographic or other imaging methods. Tanon™ High-sig ECL Western Blotting Substrate is a highly sensitive nonradioactive, enhanced luminol-based chemiluminescent substrate for the detection of horseradish peroxidase (HRP) on immunoblots. Tanon™ High-sig ECL Western Blotting Substrate Shaker / Oscillator / Mixer / Roller / Rotator.
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