b'are the two enzymes used most extensively as labelsrequires imaging instrumentation capable of capturing the for chemiluminescent protein detection. An array offluorescent signal, and the sensitivity can be lower than chromogenic and chemiluminescent substrates is availablewhat can be obtained with a well-optimized enzymatic for use with either enzyme (see pages 4259). chemiluminescent system. Although not as sensitive as enzymatic detection, fluorescent detection methods reduce AP catalyzes the hydrolysis of phosphate groupschemical waste and have the added advantage of multiplex from a substrate molecule resulting in a colored orcompatibility (leveraging multiple fluorescently tagged chemiluminescent product or the release of light as a by- antibodies to detect multiple proteins on the same blot product of the reaction. AP has optimal enzymatic activitywithout the need to strip and reprobe the blot as one would at a basic pH (pH 810) and can be inhibited by cyanides,do in a chemiluminescence-based western blot system).arsenate, inorganic phosphate, and divalent cation chelators such as EDTA. As a label for western blotting, APThe growing demand for multiplex assays has driven offers a distinct advantage over other enzymes. Becausethe development of many new fluorescent dyes and its reaction rate remains linear, detection sensitivity can beconjugation chemistries, making possible the development improved by simply allowing a reaction to proceed for aof improved fluorescent dyelabeled secondary antibodies, longer time period. such as Alexa Fluor Plus secondary antibodies. These new fluorescent secondary antibodies are brighter and more HRP catalyzes the oxidation of substrates by hydrogenphotostable than the traditional fluorophore-conjugated peroxide, resulting in a colored or chemiluminescentsecondary antibodies and comprise a broader range of product or the release of light as a by-product of thenonoverlapping spectra. Together with advances in digital reaction. HRP functions optimally at near-neutral pHimaging instrumentation, these new fluorophores provide and can be inhibited by cyanides, sulfides, and azides.a compelling reason to consider this detection strategy Antibody-HRP conjugates are superior to antibody-APversus a chemiluminescence detection strategy. conjugates with respect to the specific activities of both the enzyme and antibody. In addition, its high turnoverBiotin-binding proteins as probes rate, good stability, low cost, and wide availability ofThe highly specific affinity interaction between biotin and substrates makes HRP the enzyme of choice for mostavidin or streptavidin protein is the basis for many kinds applications. Because of the relatively small size of the HRPof detection and affinity-purification methods. Biotin is enzyme, further increases in sensitivity may be achievedvery small (244 daltons), so its covalent attachment to by using poly-HRPconjugated secondary antibodies andantibodies or other probes rarely interferes with their may eliminate the need for using ABC-type amplificationfunctions. Yet its presence as a tag on a probe allows systems, which require the use of biotinylated secondaryefficient and specific secondary detection with either avidin, antibodies, and streptavidinHRP conjugates. Streptavidin streptavidin, or Thermo Scientific NeutrAvidin Protein. HRP could bind nonspecifically to biotinylated proteins inBiotin-binding proteins are available in purified forms the sample and thereby interfere with specific detection oflabeled with enzymatic or fluorescent tags that enable the target protein.detection in many kinds of assays systems.Fluorescent labels for detectionBoth avidin and streptavidin bind very strongly and Historically, fluorophore-labeled secondary antibodies andspecifically to biotin. However, each protein has its other probes were used in a small number of cell biologylimitations in certain assays. Avidin is glycosylated, applications such as flow cytometry (FC), cell sorting, andwhich may lead to nonspecific lectin binding. Streptavidin IHC using fluorescence microscopy, but are now expandingcontains a RYD motif, a bacterial recognition sequence, into western blotting applications. The use of fluorophore- that can cause background binding with certain samples. conjugated secondary antibodies in western blottingAn alternative is to use NeutrAvidin Protein, which is an requires fewer steps compared to the use of enzymaticexclusive, deglycosylated form of avidin that avoids the labels because there is no substrate development step todrawbacks of both native avidin and streptavidin.perform. While the protocol is shorter, fluorescent detection Learn more at thermofisher.com/secondaryantibodies37'