Guidelines of Primary Antibodies for Western Blotting
Licia Miller Product Manager
Polyclonal Antibodies vs Monoclonal Antibodies vs Recombinant Antibodies
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Polyclonal, monoclonal, and recombinant antibodies are all suitable for western blotting. Polyclonal antibodies are a collection of many monoclonal antibodies that can vary between immunizations and batches. Polyclonal antibodies recognize multiple epitopes of an antigen and are therefore generally more sensitive than monoclonal antibodies that recognize only one epitope. This can be an advantage when epitope abundance, epitope masking, or epitope exposure are issues. Polyclonal antibodies are less expensive and take less time to produce.
The value of monoclonal antibodies lies in their batch-to-batch consistency and, in many cases, extensive characterization experiments and publication records. Monoclonal antibodies are usually produced by a cell line that produces a single antibody clone. When antibody production is required, these cell lines (or hybridomas) are grown in cell culture. Like any cell line that is regularly propagated, these cell lines may change over time, affecting antibody production yields and even antibody properties.
Recombinant antibodies are the best choice for achieving consistent in vitro antibody production and batch-to-batch consistency. Recombinant antibodies are produced by transfecting a production cell line with recombinant DNA encoding the desired immunoglobulin. Recombinant antibodies have several advantages: they can be modified at specific sites to add desired properties to the IgG, and they are not subject to cell line drift like hybridoma-derived monoclonal antibodies. Recombinant antibodies can be pooled to generate recombinant antibody pools, such as recombinant polyclonal primary antibodies or superclonal recombinant secondary antibodies.
Antibodies are usually provided purified in PBS or similar buffers; however, in some cases, crude antibody preparations such as serum or ascites fluid are required to maintain certain antibody properties or antibody yield. It is important to optimize the western blot protocol to minimize the impact of impurities present in crude antibody preparations on background.
Direct and indirect detection
Both direct and indirect detection methods can be used for western blotting. Each method has its own advantages and disadvantages. With direct detection, an enzyme- or fluorophore-conjugated primary antibody is used to detect the target antigen on the blot. In indirect detection, an unlabeled primary antibody is first used to bind to the antigen. Subsequently, an enzyme- or fluorophore-conjugated secondary antibody is used to detect the primary antibody. Indirect methods have many advantages over direct methods, as described below.
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Host species considerations
A variety of species can be used to generate antibodies useful in western blot applications. The most common are: mouse, rabbit, rat, goat, donkey, and chicken. The choice of host species for the primary antibody depends on whether you are probing a single target or multiple targets in a multiplex western blot experiment.
When only one antigen is being studied at a time, any host species can theoretically be used, but most primary research antibodies for western blotting are produced in immunized rabbits (polyclonal, monoclonal, recombinant) or mice (hybridoma-derived monoclonal). Some host species have additional advantages over others, for example due to their size or immunobiology. For example, when comparing mice or rabbits, rabbits are generally better able to tolerate immunization and have a much longer lifespan than mice. In addition, rabbits exhibit a more diverse natural antibody repertoire than mice, making rabbits a commonly used host for the production of polyclonal, monoclonal, and rabbit recombinant antibodies. When aiming to generate monoclonal or recombinant antibodies that are best suited for western blotting, the more repertoire of antibodies produced by rabbits, the more successfully high-affinity recombinant antibodies can be screened, isolated, and cloned. This is particularly important when aiming to make western blotting antibodies against more challenging epitopes that may not be produced in other systems.
When performing multiplex western blot experiments, use primary antibodies from different host species for each target. Ideally, use a combination of antibodies from two distantly related species, such as rat and rabbit, and avoid combinations of mouse and rat or goat and sheep. This will help in selecting the appropriate secondary antibodies to minimize potential antibody cross-reactivity, which can lead to confusing results.
Notes on Multi-Hosting Considerations
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Western blot validated antibodies
Although antibodies are designed to recognize specific target antigens, they may not work equally well in all applications. Choose an antibody that is specifically designed for western blotting or lists western blotting as an application. Additionally, it is important to confirm that the antibody is specific for native or denatured protein to determine whether SDS-PAGE or native PAGE should be performed.
When choosing a primary antibody, please confirm:
1. Antibodies validated by Western blot
2. Antibody specificity for native or denatured proteins
Aladdin antibodies undergo a rigorous two-part testing method. Part 1: Target specificity validation, Part 2: Functional application validation. Target specificity validation helps ensure that the antibody binds to the correct target , while functional application validation tests whether the antibody produces acceptable results in a specific application . Both parts of the results can be viewed directly on the Aladdin official website.
● Loading controls
Finding the right loading controls
The selection of a loading control or housekeeping protein is an important aspect of western blot normalization. For various reasons, not all loading controls can be used equally for normalization studies in all biological test systems. For example, if a chemiluminescent or single-color fluorescent system is used for target detection, the housekeeping protein should not interfere with the detection of the target (e.g., should not be of a similar molecular weight). In addition, the quantitative accuracy and linear range of the loading control in the biological test system should be evaluated before use for western blot normalization. The signal obtained with the loading control should be linear over a wide concentration range so that it can be used as a reliable reference for normalization.
For more product details, please visit Aladdin Scientific website.