Overview
Comparison of chemiluminescent substrate sensitivity. A, five different western blots were loaded with the same protein loads of purified alcohol dehydrogenase (ADH). Blots were incubated with substrates per manufacturer recommendations, then imaged for 10 sec on the ChemiDoc™ MP Imaging System. B, the signal-to-noise ratio for each band is depicted in bar graph form. Clarity and ECL Plus provided the best signal-to-noise ratios across all protein loads.
Comparison of chemiluminescent substrate signal intensity. A, five different western blots were loaded with the same protein loads of purified IKB alpha. Blots were incubated with substrates per manufacturer recommendations, then imaged for 70 sec on the ChemiDoc Touch Imaging System. B, the signal-to-noise ratios for the bands in the boxed area for Clarity Max, ECL Select, and SuperSignal West Femto are depicted in bar graph form. Clarity Max and ECL Select provided the best signal-to-noise ratios across all protein loads. ND, not determined; signal at 30 pg for SuperSignal West Femto was not detectable above background noise.
Chemiluminescence detection. The secondary antibody, which binds the antibody specific for the protein of interest, is conjugated to the horseradish peroxidase (HRP) enzyme. The HRP enzyme acts on a substrate such as luminol, releasing light. In Enhanced Chemiluminescence detection, enhancers are added to the substrate solution, greatly increasing the amount of light emitted and the duration the signal is generated. This greatly increases the sensitivity on western blots.
