b'DetectI ntroduction to western blot detection considerationsWestern blots detect specific protein from cells or tissues in a convenient, flexible format for rapid evaluation. Detectable The western blot format can also be quantitative and offerSubstrate producta high degree of sensitivity. With a variety of detection techniques, including chemiluminescent, fluorescent, or chromogenic to choose from, you can select a technologyAntibodies Enzymeto match your experimental requirements and the instruments you have available. We discuss below a few key factors to consider before performing western blotting.Direct detection Indirect directionAdvantages: Advantages:Quicker since only one antibodySecondary antibodies can Signal-to-noise ratio is used amplify signalNo concern over cross-reactivity of aA variety of labeled secondary secondary antibody antibodies are availableSignal-to-noise ratio compares the level of desired or Disadvantages: One secondary antibody may be relevant signal to the level of background noise or irrelevantLabeling may reduceused with many primary antibodiessignal; the higher the ratio, the better the result. In westernimmunoreactivity of primary antibody Use of a labeled secondary antibody does not affect primary antibody blotting, the signal is the density of the specific probedPotentially high background ifimmunoreactivitythe primary antibody is not highly protein band of interest; the noise is the density of thespecific for the target protein or ifChanging the secondary antibody the antibody cross-reacts with theallows a change of detection methodbackground. In western blotting applications, optimizationblocking proteinof the signal-to-noise ratio is often more important thanLabeled primary antibodiesDisadvantages:Secondary antibodies may produce increasing the sensitivity of the system. The sensitivity ofare expensive nonspecific signals due to cross-Low flexibility in choice of primaryreactivitythe system is irrelevant if the signal cannot be adequatelyantibody labelAdditional steps required compared distinguished from the noise. For information on westernLittle signal amplification to the direct methodblot optimization methods, see page 80. Figure 13. Comparison of direct and indirect western blot detection methods.Direct vs. indirect detectionThe antibody that recognizes a target protein is called theIndirect methods can offer increased sensitivity through primary antibody. If this antibody is labeled with a tag forthe signal amplification that occurs as multiple secondary visualization purposes (typically an enzyme or fluorophore),antibody molecules bind to a single primary antibody. In direct detection of the target is possible. Typically, theaddition, a given secondary antibody will recognize most primary antibody is not labeled for direct detection.primary antibodies of the same isotype and target species, Instead a secondary antibody that has been labeled withmaking it a more versatile reagent than individually labeled a detectable tag is used to probe for the primary antibody,primary antibodies.which is bound to the target. Thus, the target is detected indirectly. Indirect detection with secondary antibodiesSeveral variants of these probing and detection strategies requires more steps than direct detection, but it can alsoexist. However, each variant depends on a specific offer significant advantages over using primary antibodiesprobe (e.g., a primary antibody) whose presence is linked that are directly labeled (Figure 13). directly or indirectly to some sort of measurable tag. In this handbook, most methods discussed use indirect detection, as this has emerged as the most popular detection strategy.28'