Selection of Antibodies: 10 Steps

From protein immunoblotting to immunofluorescence, and then to FRET, antibodies are often an important determinant of successful and repeatable experiments. But we've never had a set "plan" to find the most suitable antibody. In this issue, the editor shares a 10-step "plan" for antibody selection.

 

Determine the Name of the Target Protein

First, you need to be clear about what protein you need to detect in order to select the corresponding antibody. In this process, you need to pay attention to the protein's Chinese and English names, aliases, and other information. You can use GeneNames to find the official name of the target protein, and then use GeneCards to identify aliases to expand the possible search range, because antibody suppliers may use different aliases to name the same protein.

 

Determine Other Information of the Target Protein

Determine the isotypes, functional domains, processed forms, structural domains with different subcellular localization (such as extracellular and intracellular, etc.) and the expected specificity of post-translational modifications of the target protein.

 

Find the Sequence of the Target Protein

Use Uniprot to find the amino acid sequence of the target protein (and its subtypes).

 

Determine Potential Cross-Reactivity With Other Species or Proteins

Use the BLAST tool in NCBI to analyze sequences, to determine whether there are different regions in the target protein with linear epitopes unique to a single antigen, and whether antibodies in the target antigen may cross-react with other proteins that have similar sequences.

 

Epitope Selection

Depending on the research purpose, choose the appropriate epitope. This epitope can be a unique area in the target antigen with specificity, allowing the antibody to accurately recognize and bind to it; or it can be those areas with cross-reactivity, allowing the antibody to interact with other proteins with similar sequences.

 

Determine the Type of Experiment

The types of experiments suitable for antibodies are often related to the type of antigenic epitope. Linear epitopes are suitable for use in protein blotting (Western Blot) and paraffin-embedded immunohistochemistry experiments, and native epitopes are more suitable for immunoprecipitation (IP), cryosection immunohistology, flow cytometry, and enzyme-linked immunosorbent assay (ELISA). In addition, those antibodies that can identify formalin-resistant epitopes and are used for immunohistochemical detection may also show good utility in other technologies using formalin fixation (such as ChIP).

 

Determine the Isotype and Host of the Primary Antibody

In a single experiment, if you need to use multiple primary antibodies for detection at the same time, it is more advantageous to choose primary antibodies with different hosts or isotypes. In this way, we can use secondary antibodies with different fluorescent labels of the same isotype or host specificity to detect multiple targets at the same time. This method is particularly common in colocalization or protein interaction studies.

 

Selection of Antibody Clonality

The selection of antibody clonality depends on its expected use. Polyclonal antibodies can recognize multiple epitopes on the antigen, the detection signal is strong, but the specificity is relatively low, so polyclonal antibodies use technologies such as Western Blot to detect antigens by distinguishing non-target binding bands at different molecular weights. In contrast, monoclonal antibodies have higher specificity because they target only a single epitope and isotype, making them perform well in research on natural tissues, such as IP and flow cytometry applications.

 

Literature Search

Once the required antigen and antibody characteristics are determined, the next step is to conduct an in-depth literature search to find publicly published antibody information in similar experimental environments (such as the same application, tissue type or cell line). Tools like BenchSci can be used. When reviewing the literature, carefully review the usage data of the antibodies, especially those antibodies with inconsistent effects. If the paper does not provide validation data, you can contact the author to request relevant information.

 

Determine the Priority of Candidate Antibodies and Validate

After a literature search, list the antibodies for sale on the market. Prioritize antibodies according to the product data sheet, making sure they match the antigen and antibody characteristics in steps 1-8. Before applying the antibodies to research, be sure to perform validation experiments (such as knockout experiments, immunoprecipitation mass spectrometry or CRISPR).