Choosing the correct antibodies is a determining step in the success or otherwise of any immunoassay. That is why we present below a series of important guidelines to take into account when selecting the appropriate antibodies to successfully carry out your tests.
Antibody datasheets detail the applications or techniques in which they have been successfully tested. Additionally, when an antibody is tested in an application and the desired results are not obtained, this information is usually also included to prevent researchers who may be interested in using it in the same way. On the other hand, if an application is not included in the list of tested techniques, this means that the antibody in question has not been tested in that technique and that its performance in that application is not known with certainty.
Antibodies available to the public are constantly undergoing various tests and controls and the respective data sheets are updated with information regarding compatible applications. For further assistance on this topic you can download the Abcam Protocol Book.
NATURE OF THE SAMPLE
The nature of the sample under study will determine which is the appropriate antibody for each case. In this sense, the following aspects must be taken into account:
The region of the protein to be detected: Antibodies are generated by immunizing host animals with an immunogenic substance, these substances may be known as immunogens, complete proteins, fragments of proteins, peptides, complete organisms (for example, bacteria) or cells. . The immunogen is generally described in the data sheet, although in some cases an exact description is not provided for reasons of patent rights. Either way, it is important to verify that the immunogen is identical or is contained within the region of the protein that is being detected. For example, if you are trying to detect a cell surface protein in living cells using Fluorescence-activated cell sorting (FACS), you should choose an antibody generated against an extracellular domain of the protein of interest.
Note: FACS is a specialized type of flow cytometry that allows a heterogeneous mixture of cells to be classified into two or more vessels, one cell at a time, based on the specific light scattering and fluorescent characteristics of each cell.
Sample Processing: Some antibodies require that samples be treated specifically. Many antibodies will only recognize proteins that have been reduced and denatured, because this reveals epitopes that would otherwise be hampered by the secondary and tertiary structure of the proteins. On the other hand, some antibodies will only recognize epitopes on proteins in their native folded state. For example, Abcam’s antibodies to Western Blot require samples to be reduced and denatured unless otherwise stated in the data sheet.
For immunohistochemistry, some antibodies are only appropriate for unfixed frozen tissues, while others cannot bind their targets in formalin-fixed and paraffin-embedded tissues without a prior antigen recovery step that reverses the crosslinks introduced by formalin-binding .
If possible, it is advisable to choose an antibody that has been generated against the same species from which the study sample comes. The antibody can react with the same target protein from other species that share sufficient amino acid sequence homology. If the sample is not from one of the species mentioned in the data sheet, this means that it has not been analyzed and its suitability cannot be demonstrated. Many times a cross-reactivity prediction based on sequence similarity is made and this information is placed on the data sheet, except that it is only a prediction.
CHOICE OF THE PRIMARY ANTIBODY HOSPITAL SPECIES
The species in which a primary antibody is generated must be different from the species of the study sample, this in order to avoid cross-reactivity of the secondary anti-immunoglobulin antibody with the endogenous immunoglobulins in the sample. For example, if you are studying a mouse protein, it is advisable to choose a primary antibody generated in a species other than the mouse. A primary antibody produced in rabbit would be an appropriate choice in this example, followed by a secondary anti-rabbit IgG antibody.
For techniques using samples that do not contain endogenous immunoglobulin (IgG), the choice of the host species of the primary antibody is less critical. An example is the Western blotting of a cell lysate that is not expected to contain IgG. However, tissue lysates and serum-containing tissue culture supernatants will contain immunoglobulins. IgG will appear on Western blots of reduced denatured samples as 50 and 25 kDa bands corresponding to the heavy and light chains of the IgG molecule.
CHOOSING THE RIGHT SECONDARY ANTIBODY
Secondary antibodies should be selected against the host species of the primary antibody being used. For example, if the primary is a mouse-generated monoclonal, an anti-mouse secondary will be required. It is recommended that you consult the secondary antibody data sheet to ensure that it has been tested in the application to be used. This topic will be discussed in depth in a future article.
CHOICE OF ANTIBODIES FOR DOUBLE STAINING
Double immunostaining of cell cultures or tissues requires that primary antibodies are generated in different species and that secondary antibodies recognize one of the species exclusively. Abcam offers a wide range of secondary anti-Ig antibodies that have been pre-absorbed against immunoglobulins from other species to eliminate cross-reactivity. Alternatively they also offer directly conjugated primary antibodies that eliminate the need for secondary antibodies.
CHOICE OF THE RIGHT CONJUGATION WITH FLUOROCROMES AND CHROMOGENS
Certain molecules, commonly known as tags, are conjugated to antibodies to visualize the presence of the target protein. Choosing the right label depends on the application to be used:
- Fluorescent labels emit light in the visual range when excited by light of a specific wavelength. Several are available, all with their own excitation and emission characteristics.
- The horseradish peroxidase (HRP) and alkaline phosphatase (AP) enzyme labels form a colored precipitate when combined with the appropriate substrate.
- Biotinylated antibodies are useful for signal amplification when followed by an avidin-biotin-enzyme or fluorochrome complex (commonly abbreviated as ABC reagent), or avidin or streptavidin conjugated with an enzyme or fluorochrome.