How to choose antibodies wisely?
How to choose antibodies wisely?
There are a variety of antibodies available for detecting target proteins for any purpose. In order to narrow down the choice of antibodies and select the appropriate one, the following factors need to be considered:
1) Reference
2) Antigenic epitope
3) Application of the antibody
4) Species of antibody and sample
5) Selection of mono- and poly-antibodies
1. References
Check which antibodies have been used by other researchers to study your target protein of interest by reviewing the literature, and prioritize these antibodies if their scope of application is consistent with your experimental application.
2. Antigenic epitope
If you are studying a specific region of the target protein, such as a specific phosphorylation site, you can verify the antibody immunogen information and select the antibody whose immunogen is consistent with the target region you are studying.
3. Application range of the antibody
Generally, the specification of the antibody will list the types of experiments for which the antibody is validated (e.g., WB, IHC, IP, IF, etc.). If the antibody specification does not mention the types of applications, it does not mean that the antibody is not suitable for such types of analytical applications, but only indicates that the antibody has not yet been validated by such experiments, so try your best to select the antibody suitable for your experiments according to the validated types listed in the specification.
4. Antibody and Sample Genus
Determine the genus of the sample, e.g., Human, Mouse, Rat, etc. Generally, the antibody's specification will list the genus of the antibody. Generally, the instruction manual of the antibody will list the species for which the antibody is suitable for the experiment, please select the antibody suitable for your experiment according to the verified species listed in the instruction manual of the antibody.
5. Selection of monoclonal and polyclonal antibodies
Generally speaking, monoclonal antibody recognizes a single antigenic epitope, which has high specificity but relatively low affinity and low sensitivity in detecting antigens; whereas polyclonal antibody recognizes multiple antigenic epitopes on the target proteins, which has a slightly weaker specificity, and sometimes cross-reacts with other proteins, but the antibody's affinity is strong and its sensitivity is high. In flow cytometry experiments, monoclonal antibodies are more effective because polyclonal antibodies recognize too many antigenic epitopes, which will lead to inaccurate data. in IP experiments, however, polyclonal antibodies can obtain satisfactory results.