The antibodies are often times a determining factor for success and reproducibility of the tests. Today’s large commercial supply of antibodies makes it not always easy to choose the one that works well in a specific application .
Many of you frequently ask us about the key aspects to look for in selecting the most suitable antibody . So today we bring you this short guide that we hope will help you choose an antibody that best suits your assay by following these 10 easy steps.
1.- Identify the target protein and all its alternative names , since different manufacturers of antibodies can refer to them based on their different aliases.
2.- Define the antigenic restrictions , that is, define if you need the antibody to be specific for a specific isoform, a functional domain, a specific fragment or a specific post-translational modification.
3.- Obtain the sequence of the target protein using a tool such as UNIPROT .
4.- Identify the potential cross reactivity with other species and / or proteins . To do this, you can analyze the sequence of the target protein with the BLAST tool from NCBI.
5.- Define the ideal epitope depending on the objective of the study; The one that confers the most specificity is not always sought.
6.- Define the techniques and applications in which you will use the antibody. This point is very important, since the antibodies may not work the same when the protein is in its native form (which occurs in ELISA, Flow Cytometry or Immunoprecipitation immunoassays, among others), as in denatured form (Western Blot, IHC in paraffin, etc.).
7.- Decide the isotype and the “host” for the primary antibody. The isotype may vary depending on the secondary antibody that we are going to use. Regarding the “host”, we remind you of this recent entry on Species for the production of antibodies .
8.- Decide the type of antibody , monoclonal or polyclonal, depending on the type of assay and its purpose. As a general rule, polyclonal antibodies are more sensitive and monoclonal antibodies more specific and homogeneous between batches.
You can expand the information on the differences between monoclonal and polyclonal antibodies here .
9.- Look for commercial options that fit the specifications of the previous points: check bibliographic references, validation data, manufacturer reliability, etc.
10.- And if after the first 9 points you still have doubts, contact us! We will be happy to advise you on the best option for your essay.