Potential for non-specific binding that may increase background in certain samples.Selection of directly conjugated primary antibodies is limited.Label may interfere with target binding, resulting in lower sensitivity and higher background.Further signal amplification possible with biotinylated secondaries and fluorescent or enzyme-labeled streptavidin.Save time with multiplexed detection using fluorescent secondary antibodies.Many options using HRP, Alexa Fluor or Alexa Fluor Plus labeled secondary antibodies.Signal amplification, since multiple labeled secondaries bind to each primary.Eliminates possible background by secondary antibody cross-reactivity in certain samples.Saves time by eliminating secondary antibody incubation step.Unlabeled primary antibody is detected using an enzyme- or fluorophore-conjugated secondary antibody Primary antibody is conjugated with an enzyme or fluorescent dye for direct detection. It is important to optimize western blotting protocols to minimize the impact of impurities present in crude antibody preparations on background. ![]() Recombinant antibodies can be pooled to generate recombinant antibody pools, such as recombinant polyclonal primary antibodies or superclonal recombinant secondary antibodies.Īntibodies are usually provided purified in PBS or similar buffers however in some cases, crude antibody preparations such as serum or ascites fluid are necessary in order to maintain certain antibody characteristics or antibody yield. Recombinant antibodies have several benefits: They can be modified at specific sites to add desired characteristics to IgGs and they are not subject to cell line drift such as hybridoma derived monoclonals. Recombinant antibodies are produced by transfecting production cell lines with recombinant DNA that encodes the desired immunoglobulins. Recombinant antibodies are the best option for consistent, animal origin-free antibody production and lot-to-lot consistency. Just like any often-propagated cell line, these cell lines could potentially undergo gradual changes affecting antibody production yields or even antibody characteristics. These cell lines (or hybridomas) are grown in cell culture when the antibody is needed for production. Monoclonal antibodies are usually produced by cell lines that generate one individual antibody clone. Monoclonal antibodies are valued for their lot-to-lot consistency and in many cases, extensive characterization and publication history. Polyclonal antibodies are less expensive and less time-consuming to produce. This can be a benefit when epitope abundance, epitope masking or epitope exposure is a concern. Polyclonal antibodies recognize multiple epitopes of an antigen and are therefore usually more sensitive than monoclonal antibodies that recognize only one epitope. Polyclonal antibodies are a pool of many monoclonal antibodies, which can vary from immunization to immunization and lot-to-lot. Polyclonal, monoclonal and recombinant antibodies all work well for western blotting. Cell line drift could result in subtle, long-term changes to antibody. Sensitivity of detection is dependent on abundance and exposure of a single epitope. Epitopes similar to target can contribute to detection of unspecific bands. Lot-to-lot variability of antibody pool can result in inconsistent detection. Stable, long-term supply with lot-to-lot consistency. Often well characterized, historic knowledge of specific clones, publications for performance in western blotting. ![]() Many antibodies in the polyclonal pool can bind epitopes on antibody target Can be modified on the DNA level or used to generate defined antibody pool. Single antibody derived from recombinant DNA. Single antibody produced by identical B cell clones that recognize one epitope on the same antigen Collection of antibodies from different B cells that recognized multiple epitopes on the same antigen
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