Primary antibody selection considerations for western blot analysis
Polyclonal vs monoclonal vs recombinant antibodies
| Polyclonal | Monoclonal | Recombinant | |
|---|---|---|---|
| Definition | Collection of antibodies from different B cells that recognized multiple epitopes on the same antigen | Single antibody produced by identical B cell clones that recognize one epitope on the same antigen | Single antibody derived from recombinant DNA. Can be modified on the DNA level or used to generate defined antibody pool. |
| Advantages | Highly sensitive. Many antibodies in the polyclonal pool can bind epitopes on antibody target | Lot-to-lot consistency. Often well characterized, historic knowledge of specific clones, publications for performance in western blotting. | Stable, long-term supply with lot-to-lot consistency. Not susceptible to cell-line drift. |
| Disadvantages | Lot-to-lot variability of antibody pool can result in inconsistent detection. Epitopes similar to target can contribute to detection of unspecific bands. | Sensitivity of detection is dependent on abundance and exposure of a single epitope. Cell line drift could result in subtle, long-term changes to antibody. | Very specialized and epitope dependent. Longer development time. May need more upfront optimization. Usually higher price. |
Polyclonal, monoclonal and recombinant antibodies all work well for western blotting. Polyclonal antibodies are a pool of many monoclonal antibodies, which can vary from immunization to immunization and lot-to-lot. Polyclonal antibodies recognize multiple epitopes of an antigen and are therefore usually more sensitive than monoclonal antibodies that recognize only one epitope. This can be a benefit when epitope abundance, epitope masking or epitope exposure is a concern. Polyclonal antibodies are less expensive and less time-consuming to produce. Monoclonal antibodies are valued for their lot-to-lot consistency and in many cases, extensive characterization and publication history. Monoclonal antibodies are usually produced by cell lines that generate one individual antibody clone. These cell lines (or hybridomas) are grown in cell culture when the antibody is needed for production. Just like any often-propagated cell line, these cell lines could potentially undergo gradual changes affecting antibody production yields or even antibody characteristics. Recombinant antibodies are the best option for consistent, in vitro-derived antibody production and lot-to-lot consistency. Recombinant antibodies are produced by transfecting production cell lines with recombinant DNA that encodes the desired immunoglobulins. 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 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 necessary in order to maintain certain antibody characteristics or antibody yield. It is important to optimize western blotting protocols to minimize the impact of impurities present in crude antibody preparations on background.
Direct vs indirect detection
Both direct and indirect methods of detection can be used in western blotting. Each method provides its own advantages and disadvantages. With the direct detection method, an enzyme- or fluorophore-conjugated primary antibody is used to detect the antigen of interest on the blot. In the indirect detection method, an unlabeled primary antibody is first used to bind to the antigen. Subsequently, the primary antibody is detected using an enzyme- or fluorophore-conjugated secondary antibody. The indirect method offers many advantages over the direct method, which are described below.
| Direct method | Indirect method | |
|---|---|---|
| Description | Primary antibody is conjugated with an enzyme or fluorescent dye for direct detection. | Unlabeled primary antibody is detected using an enzyme- or fluorophore-conjugated secondary antibody |
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Host species considerations
Multiple species are used to generate antibodies that can be used in western blot applications. Most commonly: mouse, rabbit, rat, goat, donkey and chicken. Which host species primary antibody to choose will depend on whether a single target is being probed or multiple targets are being probed in a multiplex western blot experiment. When investigating only one antigen at a time, theoretically any host species can be used, however most primary research antibodies for western blotting are produced from immunized rabbits (polyclonal, monoclonal, recombinant) or mice (hybridoma derived monoclonals). Some host species provide additional advantages over others for example due to their size or immune biology. When comparing mouse or rabbit for example, rabbits usually are better at tolerating immunizations and have a significantly longer life span than mice. Furthermore, rabbits exhibit a more diverse natural repertoire of antibodies than mice, which makes rabbits a popular host for the generation of polyclonal, monoclonal and rabbit recombinant antibodies. When aiming to generate the most suitable monoclonal or recombinant antibody for western blotting, the greater repertoire of rabbit-produced antibodies allows for more successful screening, isolation and cloning of high affinity recombinant antibodies. This is especially important when aiming to make western blot antibodies to more challenging epitopes that may not be feasible to produce with other systems.
When performing a multiplex western blot, use primary antibodies from different host species for each target being probed. Ideally, use a combination of antibodies from two distantly related species such as rat and rabbit, avoiding combinations like mouse and rat or goat and sheep. This will aid in the selection of appropriate secondary antibodies to minimize potential antibody cross-reactivity, which can lead to confusing results.
Multiplexing host considerations
| First antibody host species | Secondary antibody host species | |
|---|---|---|
| Rat | Rabbit | ✓ |
| Mouse | Rabbit | ✓ |
| Goat | Rabbit or mouse | ✓ |
| Mouse | Rat | X |
| Goat | Sheep | X |
Western blot validated antibodies
Although antibodies are designed to recognize a specific target antigen, they may not work equally in all applications. Choose antibodies designated specifically for western blotting or that list western blotting as an application. In addition, it is important to confirm that the antibody is specific towards the native or denatured protein, to determine if SDS-PAGE or native PAGE should be performed.
When selecting a primary antibody, confirm:
- Antibody is validated for western blotting
- Antibody specificity towards the native or denatured protein
Invitrogen antibodies undergo a rigorous 2-part testing approach. Part 1: Target specificity verification, Part 2: Functional application validation. Target specificity verification helps ensure the antibody will bind to the correct target. Most antibodies were developed with specific applications in mind. Testing that an antibody generates acceptable results in a specific application is the second part of confirming antibody performance. Learn more about Invitrogen Antibody Validation process.
