Choosing a Primary Antibody

Primary antibodies are essential tools in biological research aiding the detection and identification of protein targets within cells and tissues. Selecting the right primary antibody is critical to obtaining reliable and reproducible results.

When selecting a primary antibody, there are several factors to consider.

Clonality refers to whether the antibodies come from different B-cells (polyclonal antibodies) or identical B-cells derived from a parent clone (monoclonal antibodies). Each type of antibody has its own advantages and limitations.

  • Polyclonal antibodies are a heterogeneous mixture of antibodies that recognize different epitopes of a specific antigen. They have higher overall antibody affinity against the antigen due to the recognition of multiple epitopes resulting in more robust signal and high sensitivity for detecting low-abundance proteins. Limitations may include limited supply, lot-to-lot variability, and cross-reactivity. High quality suppliers such as Aves Labs and PhosphoSolutions have developed unique techniques such as our serum pooling practice to provide polyclonal antibodies which are highly validated and deliver lot-to-lot consistency.
  • Monoclonal antibodies are a homogenous population of antibodies that only recognize a single epitope per antigen. They have high specificity for their target, low non-specific cross-reactivity, and minimal batch-to-batch variation. Limitations may include a lack of versatility across applications, species, and conformational changes in proteins due to their single binding site.
  • Recombinant antibodies are produced in vitro using synthetic genes. They offer a long-term, secured supply with minimal batch-to-batch variation. The antibody-encoding sequence is known and defined, allowing for further engineering and manipulation for specific uses.

 

It is at the discretion of the scientist to determine which type of antibody is best suited and optimized for their applications. Monoclonal antibodies are more desirable in settings such as diagnostic manufacturing where large amounts of identical antibody that bind to a single epitope are required. In other applications such as analyzing low-abundance targets or detecting post-translational modifications, polyclonal antibodies are better suited. Whether monoclonal or polyclonal, it is critical to source your reagent from trustworthy suppliers such as Antibodies Inc.

Related articles: The History of Phospho Polyclonal Antibodies

Antibody validation in specific applications and species

When choosing a primary antibody, it is important to ensure that it has been validated to bind the target. The antibody datasheets should provide information on the applications and species in which the antibody has been successfully tested.

If the datasheet does not provide information on a particular application and species, it is uncertain how the antibody will perform in that specific context. In such cases, it is recommended to contact techsupport@antibodiesinc.com to discuss further.

Antibody specificity confirmed by knock-out validation

A high-quality antibody demonstrates target specificity, enabling it to detect the protein of interest even when it is expressed at low levels. However, numerous studies have indicated that not all antibodies possess this level of specificity, as many exhibit cross-reactivity with non-target proteins.

Knock-out (KO) validation is widely recognized as one of the most reliable and trusted methods for validating antibody specificity. This robust technique involves testing the antibody in a KO cell line, cell lysate, or tissue that lacks expression of the target protein. A specific antibody should not produce any signal in the KO cell line, while still yielding a specific signal in the wild-type cell line. By utilizing KO validation, researchers can establish a true negative control to ensure the accuracy of their results.

Immunofluorescence staining of adult wild-type (WT) and Kv4.2 knockout (Kv4.2-/-) mouse hippocampus with K57/1 (red) and Kv2.1 rabbit (green).

Immunogen Information

Antibody discovery involves immunizing host animals with an immunogen which, take the form of full-length proteins, peptides, or whole cells. The datasheet usually provides information about the immunogen, although the immunogen sequence may not be available if it is proprietary.

The choice of immunogen will determine which region of the protein the antibody binds to. If the immunogen sequence is available, it is important to ensure that it is either identical to or contained within the region of the protein you are trying to detect. For instance, if you are attempting to detect the intracellular part of a protein in live cells, it is advisable to select an antibody that has been raised against the intracellular domain.

Carefully Select Host Species

If you plan to perform indirect detection using secondary antibodies, it is advisable to select a primary antibody that is raised in a different species than your sample. This will help prevent any cross-reactivity between the secondary antibody and the endogenous immunoglobulins present in the sample. For example, if you are studying a mouse protein, it is recommended to choose a primary antibody raised in a species other than a mouse, such as a rabbit or chicken. Cross-reactivity is a concern when working with tissue samples, but not with cell lines, as it arises from the presence of host antibodies in the sample.

However, if you are required to use a primary antibody with the same host species as your tissue sample, you will need to carefully modify your protocol to minimize background staining. Alternatively, you can consider using chimeric antibodies that consist of domains from different species to avoid cross-reactivity.

In certain cases, when working with non-model organisms that are not commonly used in research, you may need to use an antibody that has not been tested in your species. In such situations, it is important to check the alignment of the antibody's immunogen sequence with your protein of interest. You can find antibody immunogen sequences using the UniProt/SwissProt protein database link provided on online datasheets. By comparing the immunogen sequence with your protein using tools like CLUSTALW, an alignment score of over 85% indicates that the antibody may bind to your protein. However, it is important to note that this does not guarantee optimal performance, and further experimentation will be necessary.

Processing of Samples

An antibody specifically recognizes an epitope in a specific conformation which may be altered during sample processing, such as through fixation and cross-linking using formaldehyde. As a result, certain antibodies only function effectively on samples that have been processed in a particular manner. Some antibodies only recognize proteins that have been reduced and denatured, as this exposes epitopes that would otherwise be hidden. Conversely, some antibodies only recognize epitopes on proteins in their native state.

In the case of immunohistochemistry, certain antibodies are suitable only for unfixed frozen tissue, while others that have been formalin-fixed and paraffin-embedded require an antigen retrieval step to expose the epitope. It is recommended to review the antibody datasheet for any restrictions on sample processing.

Be Prepared to Validate the Antibody Yourself

Although an antibody may be well characterized and highly cited within peer-reviewed publications, it is always good practice to re-validate the antibody within your own laboratory. Therefore, it is essential to use the correct tissue types and controls to monitor specificity and sensitivity. Pre-validation of the antibody within your laboratory will ensure you have confidence in the product, and therefore future results generated using the antibody reagent.

To help you choose the right primary antibodies, here is a checklist to consider:

  1. Review datasheet to determine:
    • Antibody clonality
    • Applications
    • Species reactivity
    • Host species
    • Immunogen
  2. Analyze the images and data provided. Do the results reflect what has been published in the scientific literature?

Related articles

Antibody background information
Applications
Antibody Reagents
Accessory Reagents