How to choose the right antibody for a successful assay

Antibodies are an essential detection tool for biochemistry, cell biology and immunology laboratories. It is sometimes a daunting task to find the right one, given the large variety of antibodies and antibody fragments used for a myriad of applications, not to mention the wide range of problems that can jeopardize antibody-dependent experiments. Common causes such as cross-reactivity, batch-to-batch variability, and the use of antibodies in untested applications have been blamed for inconsistent results. Here, we share some lab-to-lab tips to select the antibody that most suits your research purposes.

Define your protein target – Understand the biology of your protein target, as protein targets can be highly complex and sequence identity, or homology, with related proteins may give potential cross-reactivity. Use databases such as GeneCards, to identify the nomenclature and any alternative names of the antigen, and UniProt to obtain the amino acid sequence of your protein of interest.

Choose the right application – Select the application that most matches your research. This depends on the type of data you need to answer your research question. For example, Western blot is the well-known application for studying protein concentration; immunohistochemistry and immunocytochemistry are used for studying localization as they work with fixed cells and tissues; while ELISA, FACS and flow cytometry are appropriate techniques for studying protein interactions and expression.

a.png

Ensure the antibody suits your sample – Because targets vary in their sequence and structure from species to species, use antibodies that have been validated specifically for your species of interest. In this case, trust the datasheet: if the antibody has been validated for one species, there is no guarantee that it will work in others.

Choose a compatible antibody – It shouldn't be assumed that an antibody that has been shown to detect a target in one application will do so in others. Immunochemistry applications depend on the use of sensitive and highly specific antibodies. More specifically, they depend on the ability of an antigen to recognize the target protein. For an efficient antigen-antibody interaction, the epitope must be readily available for binding. Sometimes the antigen may require the target protein to be present in its native and folded form, or in its denatured form.

Through fixation, pH changes, or during preparation for gel electrophoresis, the epitope may be altered, and this could affect its ability to interact with an antibody. For example, an antibody may recognize an epitope within a frozen tissue section in IHC study, but this epitope may not be accessible in a sample that has been denatured for Western blot analysis.

b.png

Select your host species – Usually, it is recommended to avoid host antibody species that are identical to the species of your target samples in order to avoid interference with immunoglobulins. When choosing a host species, consider the secondary antibodies raised against the host species of the primary antibody and selected according to the application.

Study the product datasheet and be ready to optimize your protocol – Product datasheets typically contain many important details, including the description of the immunogen, the nature of the epitope, the host species, the cross-reactivity, and recommended starting dilutions. Therefore, there are many factors to consider when setting up a protocol such as the blocking buffer, the antibody diluents, the length of the incubation steps, and the method of detection. All these additional details will help you to optimize your protocol for a successful assay.

Always include experiments controls – When planning an experiment, controls are the crucial part that helps to understand whether the result is a success or a failure. Most common experimental techniques require specific controls to help in the interpretation of the data. For example, Western blots should always include a loading control, while IHC and IF should always have more than one negative control to help distinguish signal from background noise.

c.png

Examples of positive controls might include a recombinant protein or a lysate prepared from a cell line that is known to produce detectable target levels; whilst negative controls might include using knockout samples or omitting the primary antibody, both of which allow background staining attributable to the secondary antibody to be detected.

In summary, although finding an antibody that works well for a specific application sometimes remains a difficult task, we hope that these tips and guidelines will provide some useful starting points for your search. If you would like to discuss these or other possible approaches to scrutinizing antibodies prior to purchase, get in touch with us!

 

Available from BMA Biomedicals:

T-4070: polyclonal rabbit anti Neuropeptide Y (human, mouse, rat), neat antiserum

T-1302: monoclonal mouse anti Cytokeratin (human), purified antiserum

T-4542: polyclonal rabbit anti Secretin (porcine), purified antiserum

 

Author: Sonia Accossato

Copyright © BMA Biomedicals 2022