Moreover, the success of this procedure has to be laboriously quality controlled each time. Furthermore, every new batch of serum contains another heterogeneous mixture of antibodies, which need to be affinity-purified on IgG columns and then depleted (by preadsorption) of nonspecific and cross-reacting antibodies. The need for a continuous supply of anti-IgG sera requires keeping, immunizing, bleeding, and eventually killing large numbers of goats, sheep, rabbits, and donkeys, which is not only costly but also a major animal welfare and ethical problem ( Shen, 2013 Reardon, 2016). The detection or immobilization of these primary antibodies is most often performed indirectly via polyclonal anti-IgG secondary antibodies. Mouse and rabbit antibodies are fundamental tools for numerous basic research techniques and medical diagnostic assays. They also enable simpler and faster immunostaining protocols, and allow multitarget localization with primary IgGs from the same species and of the same class. Their site-specific labeling with multiple fluorophores creates bright imaging reagents for confocal and superresolution microscopy with much smaller label displacement than traditional secondary antibodies. We demonstrate their superior performance in Western blotting, in both peroxidase- and fluorophore-linked form. Their recombinant nature allows fusion with affinity tags or reporter enzymes as well as efficient maleimide chemistry for fluorophore coupling. They can be produced at large scale in Escherichia coli and could thus make secondary antibody production in animals obsolete. Here, we introduce a sustainable alternative, namely nanobodies against all mouse IgG subclasses and rabbit IgG. Their animal-based production is, however, a major ethical problem. Note: Please see the IRDye 680LT antibody pack insert for specific detergents and protocol modifications needed for optimization.Polyclonal anti–immunoglobulin G (anti-IgG) secondary antibodies are essential tools for many molecular biology techniques and diagnostic tests. Other assays will require user optimization. IRDye 680LT Secondary Antibodies tend to require more optimization than IRDye 680RD Secondary Antibodies, but they have the advantage of typically being used at much lower concentrations. Choose IRDye 680LT Secondary Antibodies for applications that require greater light stability (such as microscopy) and when optimal Western blot conditions for specific targets have already been well-established.Dilution working range is 1:5,000 – 1:25,000 for Western blots and 1:200 – 1:1,200 for In-Cell Western Assays. Choose IRDye 680RD Secondary Antibodies for the lowest background and easiest optimization when detecting in the 700 nm channel.Use IRDye 800CW for detecting targets that require the highest sensitivity. Choose IRDye 800CW Secondary Antibodies for low background and high sensitivity in the 800 nm channel.Here are some general guidelines to help you choose the right secondary antibodies: ![]() Select the right IRDye Secondary Antibody for your application using information in the table below. Choosing the Right Dye-Conjugated Antibody IRDye Secondary Antibodies have been optimized for use with the Odyssey ® family of imaging systems and can be used on instruments with similar excitation and emission filters. IRDye Secondary Antibodies are highly cross-adsorbed, making them suitable for multi-color detection. Quality and performance of secondary antibodies is crucial for Western blotting, In-Cell Western ™ Assays, immunohistochemistry, and many other applications. Highly Cross-Adsorbed IRDye Secondary Antibodies Provide Superior Performance
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