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Updated August 8, 2023
A primary antibody is an antibody that binds directly to a target protein, with a variable antibody region recognizing a protein's epitope. When choosing a primary antibody, consider the following:
Clonality is determined by whether the antibodies come from different B-cells (polyclonal antibodies) or identical B-cells derived from a parent clone (monoclonal antibodies). These antibodies have distinct advantages and limitations covered here.
To recap, polyclonal antibodies consist of a heterogeneous mixture of antibodies, with each antibody recognizing different epitopes of a particular antigen. By binding to several different epitopes, polyclonal antibodies can produce a strong signal against the target antigen in their relevant application and are not biased against a single epitope. However, they are limited in supply, subject to high batch-to-batch variability, and exhibit cross-reactivity and lack of specificity.
In contrast to polyclonal antibodies, monoclonal antibodies only recognize a single epitope per antigen. Monoclonal antibodies have high specificity for their target, low non-specific cross-reactivity, and minimal batch-to-batch variations.
The term 'recombinant' refers to antibodies produced in vitro using synthetic genes. Compared to traditional monoclonal and polyclonal antibodies, recombinant antibodies offer long-term, secured supply with a minimal batch-to-batch variation. Since the antibody-encoding sequence is known and defined, it can be further engineered and manipulated for its intended use.
We recommend using recombinant monoclonal antibodies when a suitable clone exists for your particular target and application to ensure experimental reproducibility and long-term antibody supply. For applications where a polyclonal antibody would traditionally be used (eg, when analyzing low-abundance targets or detecting multiple post-translational modifications at once), recombinant multiclonal antibodies can offer an ideal solution. Recombinant multiclonal antibodies are a defined mixture of carefully selected individual recombinant monoclonal antibodies designed to recognize different epitopes on the same antigen. So, they can provide excellent sensitivity combined with superior specificity and reproducibility, only available from a recombinant antibody.
When selecting a primary antibody, ensure it's validated to bind the target. Antibody datasheets should list the applications and species in which the antibody has been successfully tested.
The datasheet will also highlight if an antibody was tested in a particular application and failed. If the datasheet doesn't list an application and species, it is unknown how the antibody will perform in this specific application and species. In this case, check if there are any reviews from customers who tested the antibody in your application and species of interest. All published customer reviews for a given product are listed under the "Customer reviews & Q&A" tab of the datasheet on our website.
Our antibodies are continuously tested, and datasheets are updated with the latest information on validated applications and species.
A good antibody exhibits target specificity, allowing it to identify the protein of interest even at low expression levels. However, many studies have shown that not all antibodies are specific in this way, with many displaying cross-reactivities with off-target proteins.
Knock-out (KO) validation is one of the most accepted and trusted validation processes for antibody specificity. This robust technique can confirm the antibody's specificity by testing it in a KO cell line, cell lysate, or tissue that does not express the target protein. A specific antibody should produce no signal in the KO cell line but give a specific signal in the wild-type cell line. In this way, KO validation serves as a true negative control.
Figure 1 below shows an example of KO validation for KI-67 antibody in immunocytochemistry (ICC), with Ki67 knock-out HAP1 cells (bottom) showing no expression of Ki67 (green).
Figure 1. ICC/immunofluorescence image of knock-out testing for Ki67 antibody in wild-type (top) and Ki67 knock-out HAP1 cells (bottom). Green is anti-Ki67 [EPR3610] (ab92742) with goat-anti-rabbit IgG (Alexa Fluor® 488) (ab150081), red is anti-alpha-tubulin [DM1A] (Alexa Fluor® 594) (ab195889), and blue is nuclear DNA labeled with DAPI.
We recommend you choose antibodies that have been validated in multiple applications, ideally using KO technologies. Alternatively, you can validate an antibody yourself using the appropriate KO cell line, KO cell lysate, or tissue.
Antibody discovery often starts by immunizing host animals with an immunogen. These immunogens can be full-length proteins, peptides, or whole cells. Usually, you can find information about the immunogen on the datasheet. However, the immunogen sequence won't be available if it's proprietary information.
The immunogen used will define which region of the protein antibody binds. If the immunogen sequence is publicly available, check that the immunogen is identical to or contained within the region of the protein you are trying to detect. For example, if you are trying to detect a cell surface protein on live cells by FACS, choose an antibody raised against the protein's extracellular domain.
An antibody is specific to an epitope in a particular conformation. Since sample processing will change epitope conformation (eg, fixation will lead to protein cross-linking by formaldehyde-induced methylene bridges), some antibodies only work on samples processed in a certain way. Many antibodies will only recognize proteins that have been reduced and denatured because this reveals epitopes that would otherwise be obscured. On the other hand, some antibodies will only recognize epitopes on proteins in their native state.
For immunohistochemistry, some antibodies are only appropriate for unfixed frozen tissue. Others that have been formalin-fixed and paraffin-embedded need an antigen retrieval step to expose the epitope. We recommend you check if the antibody datasheet lists any restrictions on sample processing.
If you intend to perform indirect detection with secondary antibodies, you should ideally choose a primary antibody raised in a different species to your sample. This allows you to avoid cross-reactivity of the secondary (anti-immunoglobulin) antibody with endogenous immunoglobulins in the sample. For instance, if you study a mouse protein, choose a primary antibody raised in a species other than a mouse – eg, rabbit. Since cross-reactivity emerges from the presence of host antibodies in the sample, it's a pitfall for tissue samples but not cell lines.
Suppose you have to use a primary antibody with the same host species as your tissue sample. In that case, you'll need to carefully consider how to modify your protocol to reduce background staining. Alternatively, to avoid cross-reactivity, you can use chimeric antibodies made up of domains from different species.
You don't need to worry about the primary antibody's host species with applications like western blot that use a cell lysate without any endogenous immunoglobulin (IgG) or direct detection experiments that use primary conjugated antibodies.
A note on non-model organisms
If you work in a non-model organism (ie, species not commonly used in research), you may need to use an antibody that hasn't been tested in your species. In many cases, the protein sequences are often conserved enough across several species and can be recognized with an antibody not validated in this species.
If there's no alternative to using a non-validated antibody, we recommend checking the antibody's immunogen sequence alignment with your protein of interest.
Typically, antibodies are stored in a phosphate-buffered saline (PBS) solution with carrier proteins like bovine serum albumin (BSA) and preservatives like glycerol and sodium azide. While these are essential components for maintaining antibody stability and preventing contamination, they can hinder the effective conjugation of labels (eg, fluorochromes, enzymes, and metals), affect live-cell systems, and even possibly interfere with highly specialized hardware setups.
During a typical conjugation reaction, BSA will compete with the primary antibody to attach to the label of interest, significantly reducing the conjugation efficiency. The presence of sodium azide in the antibody solution can be toxic to cells, limiting the antibody's use in cell culture and negatively affecting conjugation. Therefore, if you intend to conjugate your primary antibody or use it to stain live cells, we recommend choosing antibody formulations without carriers or preservatives.
Here's a helpful checklist for choosing your primary antibodies:
Secondary antibodies bind primary antibodies to allow detection, sorting, and purification of target antigens. They allow you to detect your protein of interest due to their specificity for the primary antibody species and isotype. Whether you need to use a secondary antibody depends on your antibody detection method.
The method of detecting the antigen of interest can be either direct or indirect (Fig. 2):
The choice of direct or indirect detection will often depend on the expression level of the target antigen. Direct detection is suitable for analyzing highly expressed antigens. On the contrary, indirect detection is preferable for studying poorly expressed antigens, which benefit from signal amplification provided by the secondary antibody.
Figure 2. A schematic representation of direct and indirect detection of antigens using antibodies labeled with a fluorophore.
Both methods have several benefits and limitations to consider before choosing the most appropriate method for your experiments (Table 1).
Table 1. Comparison of direct vs indirect detection methods.
Direct | Indirect | |
Time | Usually shorter as it only requires one labeling step | Using conjugated secondary antibodies results in additional steps and a longer time |
Complexity | Fewer steps in the protocol make this a more straightforward method | You need to select an appropriate secondary antibody or combinations of antibodies in multiplex experiments, which adds complexity |
Sensitivity | Signal may seem weaker compared to indirect methods because of the absence of secondary antibodies, which typically provide signal amplification | Several secondary antibodies may bind to the primary antibody resulting in an amplified signal |
Background | Non-specific binding is reduced | Samples with endogenous immunoglobulins may exhibit a high background |
Using directly conjugated primary antibodies (eg, conjugated to enzymatic or fluorescent labels) allows you to speed up and simplify the protocol, omitting the need for a secondary antibody staining step. Also, conjugated primary antibodies will enable you to minimize species cross-reactivity and eliminate any non-specific binding that may occur with secondary antibodies. Fluorescent-conjugated primary antibodies are ideal tools for multicolor experiments as they give you the flexibility to assemble the multiplex panel you need.
When choosing primary antibody conjugates, pay attention to antibody specificity. Ideally, go for recombinant monoclonal antibodies, which provide high specificity and batch-to-batch consistency.
Compared to secondary antibodies, primary conjugates don't provide signal amplification, so your protein of interest should be abundant in the sample. Abcam offers a wide range of primary recombinant antibodies directly conjugated to fluorescent labels or enzymes. If your antibody of choice is not available in a suitable conjugated format, you can use abcam's antibody conjugation kits.
To learn more about how to conjugate antibodies, refer to our antibody conjugation guide.
If you are using indirect detection, you will need to select an appropriate secondary antibody.
Secondary antibodies are generated by immunizing an animal with antibodies that act as immunogens. The secondary antibodies produced will bind to the antibody type with which the animal has been immunized.
Secondary antibodies have descriptive names that reveal the type of primary antibody they'll bind to (see Fig. 3, 4, and 5). These names include the prefix 'anti-' to denote their reactivity. For example, if an animal has been immunized with rabbit IgG, the secondary antibodies produced will bind to rabbit IgG and are referred to as anti-rabbit IgG.
When choosing a secondary antibody, you need to consider whether it will bind selectively to your primary antibody and enable you to detect the antigen, which is determined by several key factors outlined below.
The host species used to raise the secondary antibody must be different from that of the primary antibody. For example, if the primary antibody is raised in rabbit, your secondary antibody will need to be raised in an alternative species; a donkey anti-rabbit secondary antibody would be suitable.
Figure 3. Using the name of a secondary antibody to understand with which species it reacts. The secondary antibody is raised in donkey (A) and binds to rabbit antibodies (B).
The secondary antibody must bind to the isotype of the primary antibody.
Primary antibodies are typically IgG isotypes. Therefore, the secondary antibody will need to be raised against IgG. Usually, anti-IgG secondaries bind to the heavy & light chains (H&L) but can also be made to bind to other regions of the primary antibody.
Figure 4. Using the name of a secondary antibody to understand the species, isotype, and region of the primary antibody it binds. This secondary antibody binds specifically to the H&L region of rabbit IgGs (B, C, D).
Labels, such as fluorescent dyes, proteins, enzymes, and biotin, are conjugated to secondary antibodies to visualize the target protein's presence.
Figure 5. The secondary antibody label is usually indicated at the end of the antibody name. This secondary antibody is conjugated to Alexa Fluor® 488, a green fluorophore.
The conjugate choice depends on the application. Enzyme-linked secondary antibodies tend to be the most popular for ELISA or western blot applications. In contrast, there is a preference for secondary antibodies conjugated to fluorescent proteins or dyes (such as Alexa Fluor®) for flow cytometry and ICC.
Below we outline some suggested secondary antibodies for the main applications you're likely to use (Table 2).
Table 2. Choosing a secondary antibody labeled with an enzyme or fluorochrome for different applications.
Secondary antibodies | Enzyme | Fluorochrome |
IHC | HRP, HRP polymer, biotin (avidin/ streptavidin-conjugated to enzyme or fluorochrome) | Alexa Fluor®, Cy® dyes, FITC, PE |
ICC | - | Alexa Fluor®, Cy® dyes, FITC, PE |
Western Blot | HRP, AP | IRDye®, Alexa 680, Alexa 790 |
ELISA or ELISPOT | HRP, biotin (avidin/ streptavidin conjugated to enzyme or fluorochrome) | - |
Flow Cytometry or FACS | - | Alexa Fluor®, Cy® dyes, FITC, PE |
IHC = immunohistochemistry, ICC = immunocytochemistry, FACS = fluorescence-activated cell sorting, HRP = horseradish peroxidase, AP = alkaline phosphatase
When selecting a secondary antibody, you need to ensure that it won't cross-react with non-target proteins in the sample. You can minimize species cross-reactivity by using pre-adsorbed secondary antibodies and F(ab) antibody fragments.
Pre-adsorbed secondary antibodies are ideal for eliminating species cross-reactivity in multicolor experiments that simultaneously use several primary antibodies and corresponding secondary antibodies. Pre-adsorption (also called cross-adsorption) is an extra purification step introduced to increase antibody specificity. The pre-adsorption process reduces the risk of cross-reactivity between the secondary antibody and endogenous immunoglobulins present in cell and tissue samples.
Using F(ab) and (Fab')2 fragment antibody fragments, rather than whole antibodies, can eliminate non-specific binding between Fc portions of antibodies and Fc receptors on cells (such as macrophages, dendritic cells, neutrophils, NK cells, and B cells). F(ab) and F(ab')2 fragments also penetrate tissues more efficiently due to their smaller size. As fragment antibodies do not have Fc portions, they do not interfere with anti-Fc mediated antibody detection.
Double immunostaining of cell cultures or tissue requires two primary antibodies raised in different species and two secondary antibodies exclusively recognizing one species. To avoid cross-reactivity, you can choose secondary anti-IgG antibodies, which have been pre-adsorbed against immunoglobulins from other species. Alternatively, using directly conjugated primary antibodies will remove the need for secondary antibodies.