Primary and Secondary Antibody Selection

Introduction

Datasheets on antibody applications provide a list of successfully tested applications, and also note those in which the test has failed. Unlisted applications have not been tried with the antibody in question and the result is therefore unknown.

Abcam antibodies are constantly undergoing in-house testing so that the datasheets are kept up to date.

Samples

The choice of antibody depends upon the sample being analyzed. The following factors are of special importance:

Which Protein Domain is to be Detected

Antibodies for commercial use are produced by injecting animals with an immunogenic substance to stimulate an immune reaction. Such substances include complete proteins, fragmentary proteins, peptides, complete organisms such as bacteria, or cells. Whatever it is, the immunogen will be noted on the datasheet, though the description may sometimes be kept deliberately vague to protect proprietary rights.

The immunogen should match or at least be a part of the protein segment to be detected. For instance, if a protein present on the surface of live cells is to be detected by FACS the antibody used should have been generated by injecting the host animal with a domain of the part of the protein that is outside the cell.

Sample Processing

For some antibodies to react with the corresponding antigens in the sample, the latter needs prior preparation. This may include, for instance, reduction and denaturation of the protein to uncover epitopes that are hidden in the normal configuration of the protein by secondary and tertiary folding. Conversely, some antibodies recognize only native proteins in their functional configuration.

Abcam antibodies for Western blot testing always require prior reduction and denaturation of the protein sample, unless the datasheet specifically instructs to the contrary.

Again, when immunohistochemistry is performed, some antibodies are used which will only bind to tissue that has been frozen without fixation. However, other antibodies require the antigens in tissues which have been fixed using formalin and embedded in paraffin to be retrieved before the procedure, by reversing the formalin fixation-induced crosslinking.  Such preconditions are also described in the application datasheets.

Sample Species

Whenever possible, the antibody used should have been generated against the same species that the sample comes from.

If the amino acid sequence of the target protein both from the species in which the antibody was generated, and from another species, shows adequate homology, a cross-reaction may result. If the sample is not listed in the datasheet, it has not been tested and the antibody cannot be deemed appropriate. Cross-reactions are predicted only when there are similar sequences in target proteins from different species.

​Choosing the Species of Primary Antibody Host

The primary antibody should be raised in a species other than that from which the sample is taken. This prevents a cross-reaction between the secondary antibody (against the primary immunoglobulin) and any endogenous immunoglobulin already present in the sample.

To illustrate: if a mouse protein is to be detected, the primary antibody should not be raised in a mouse, rather in a rabbit, and the secondary anti-immunoglobulin antibody could be an anti-rabbit immunoglobulin G (IgG) antibody.

If the sample does not possess any endogenous immunoglobulin IgG activity, it is not so important to choose a different host species for the primary antibody. For instance, when a cell lysate is being tested by Western blotting, IgG is not typically present.

However, if the tissue is lysed, or if tissue culture supernatants that also contain serum are analyzed, immunoglobulins will be present. IgG in reduced and denatured samples can be seen on Western blotting as bands at 50 and 25 kDa, which represent the IgG heavy and light chains.

Choosing a Secondary Antibody

Any secondary antibody should act against the host animal species from which the primary antibody was raised. Thus, for a primary mouse monoclonal antibody, an anti-mouse IgG is chosen. The secondary antibody must have been tested in the type of application envisaged, as can be confirmed from the datasheet.

Choosing Antibodies for Dual Staining

Cell cultures or tissue can be doubly immunostained. This requires that the two primary antibodies be generated in different species, but the secondary antibodies should recognize one and only one of the two.

Abcam has a range of secondary anti-Ig antibodies which have been protected against cross-reactivity by a process of preadsorption using immunoglobulins raised in different species. Another method to achieve this is to use the Abcam primary antibodies, which are directly conjugated so as not to require secondary antibodies.

​Fluorochrome and Chromagen Labels

Antibodies are often conjugated with labels to make it easy to visualize the bound target protein. Labels come in different forms, depending on the application.

Fluorescent labels are those which emit visible light when they are excited by light which has a particular wavelength, and there are many available for use with their own particular excitation and emission parameters.

Examples of enzymes which are used as labels include horseradish peroxidase (HRP) and alkaline phosphatase (AP), which form a colored precipitate if the right substrate is added to the enzyme.

Amplified signals may be obtained by the use of biotinylated antibodies, succeeded by avidin-biotin-enzyme or fluorochrome complex, termed the ABC reagent. Other possibilities include enzyme- or fluorochrome-conjugated streptavidin or avidin.

About Abcam

Abcam is a global life sciences company providing highly validated antibodies and other binders and assays to the research and clinical communities to help advance the understanding of biology and causes of disease.

Abcam’s mission is to serve life scientists to help them achieve their mission faster by listening to their needs, continuously innovating and improving and by giving them the tools, data and experience they want. Abcam’s ambition is to become the most influential life science company for researchers worldwide.


Sponsored Content Policy: News-Medical.net publishes articles and related content that may be derived from sources where we have existing commercial relationships, provided such content adds value to the core editorial ethos of News-Medical.Net which is to educate and inform site visitors interested in medical research, science, medical devices and treatments.

Last updated: Jun 6, 2019 at 12:49 PM

Citations

Please use one of the following formats to cite this article in your essay, paper or report:

  • APA

    Abcam. (2019, June 06). Primary and Secondary Antibody Selection. News-Medical. Retrieved on September 17, 2019 from https://www.news-medical.net/whitepaper/20180619/Primary-and-Secondary-Antibody-Selection.aspx.

  • MLA

    Abcam. "Primary and Secondary Antibody Selection". News-Medical. 17 September 2019. <https://www.news-medical.net/whitepaper/20180619/Primary-and-Secondary-Antibody-Selection.aspx>.

  • Chicago

    Abcam. "Primary and Secondary Antibody Selection". News-Medical. https://www.news-medical.net/whitepaper/20180619/Primary-and-Secondary-Antibody-Selection.aspx. (accessed September 17, 2019).

  • Harvard

    Abcam. 2019. Primary and Secondary Antibody Selection. News-Medical, viewed 17 September 2019, https://www.news-medical.net/whitepaper/20180619/Primary-and-Secondary-Antibody-Selection.aspx.

Other White Papers by this Supplier