10 Steps for Antibody Selection
From Western blotting to immunofluorescence and then to FRET, antibodies are often a crucial factor for successful and reproducible experiments. However, we have never had a set 'protocol' for finding the most suitable antibodies. In this issue, we share a 10-step 'protocol' for selecting antibodies.
1. Identify the target protein name
First, it is necessary to clarify what protein needs to be detected to select the corresponding antibody. Pay attention to the protein's names in both English and Chinese, aliases, and other information. Use GeneNames to find the official name of the target protein, and then use GeneCards to identify aliases to broaden the potential search queries, as antibody suppliers may use different aliases for the same protein.
2. Determine other information about the target protein
Identify the isoforms, functional domains, processing forms, domains with different subcellular localizations (such as extracellular and intracellular), and expected specificity of post-translational modifications of the target protein.
3. Search for the target protein sequence
Use Uniprot to find the amino acid sequence of the target protein and its isoforms.
4. Determine potential cross-reactivity with other species or proteins
Use NCBI's BLAST tool to analyze the sequence and determine if there are regions in the target protein with unique linear epitopes specific to the antigen, and whether antibodies to the target antigen might cross-react with other proteins with similar sequences.
5. Epitope selection
Select the appropriate epitope based on the research purpose. This epitope can be a unique region in the target antigen that allows the antibody to accurately recognize and bind to it, or it can be a region with cross-reactivity, allowing the antibody to interact with other proteins with similar sequences.
6. Determine the type of experiment
The type of experiment the antibody is suitable for is often related to the type of antigen epitope. Linear epitopes are suitable for Western Blot and paraffin-embedded immunohistochemistry experiments, while native epitopes are more suitable for immunoprecipitation (IP), frozen section immunohistology, flow cytometry, and enzyme-linked immunosorbent assays (ELISA). Additionally, antibodies that can recognize formalin-resistant epitopes and are used in immunohistochemistry may also perform well in other formalin-fixed techniques (such as chromatin immunoprecipitation (ChIP)).
7. Determine the isotype and host of the primary antibody
In a single experiment, if multiple primary antibodies are required for detection, it is more advantageous to select primary antibodies with different hosts or isotypes. This way, we can use secondary antibodies specific to different isotypes or hosts labeled with different fluorophores to simultaneously detect multiple targets. This method is particularly common in co-localization or protein interaction studies.
8. Selection of antibody clonality
The choice of antibody clonality depends on its intended use. Polyclonal antibodies can recognize multiple epitopes on the antigen, resulting in stronger detection signals but relatively lower specificity. Thus, polyclonal antibodies are used in techniques like Western Blot where they can help distinguish non-target binding bands at different molecular weights to detect antigens. In contrast, monoclonal antibodies have higher specificity because they target a single epitope and isotype, making them excellent for studies in native tissues such as IP and flow cytometry applications.
9. Literature search
Once the desired antigen and antibody characteristics are determined, the next step is to conduct a thorough literature search to find published antibody information used in similar experimental conditions (such as the same application, tissue type, or cell line). Tools like BenchSci can be helpful. When reviewing literature, carefully examine the antibody usage data, especially for antibodies with inconsistent performance. If the paper does not provide validation data, consider contacting the authors for additional information.
10. Prioritize and validate candidate antibodies
After the literature search, list the antibodies available on the market. Prioritize them based on product datasheets to ensure they match the antigen and antibody characteristics identified in steps 1-8. Before applying the antibody to research, be sure to conduct validation experiments (such as knockout experiments, immunoprecipitation-mass spectrometry, or CRISPR).
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