How to choose and use antibodies

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 in Chapter 2. 

To recap, polyclonal antibodies consist of a heterogeneous mixture of antibodies, with each antibody recognizing different epitopes of a particular antigen (Fig. 5, Chapter 2). 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 (Fig. 5, Chapter 2). 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 16 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).

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.

As described in Chapter 2, 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.

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. 

• You can find antibody immunogen sequences using the UniProt/SwissProt protein database link on online datasheets. 

• Take this immunogen sequence and compare it with the protein you're interested in using an online tool like CLUSTALW. 

• An alignment score of over 85% indicates that the antibody may bind to your protein. However, this doesn't guarantee the antibody will perform well; you'll need to run several controls to ensure it works as intended.

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.

• Check antibody clonality, application suitability, host species, and species reactivity – you can find these on the datasheet.

• Keep in mind that our guarantee will cover only applications and species listed on the antibody datasheet.  

• Take a look at the images and data provided – does the data look robust?

• Read recent publications citing the antibody and customer reviews on the datasheet; these should give you a good idea of how the antibody performs.

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. 15): 

• Direct: antigen is detected by a primary antibody directly conjugated to a label (ie, conjugated primary antibody), so no secondary antibody is required.

• Indirect: antigen is detected by a conjugated secondary antibody that has been raised against the primary antibody's host species and binds to the primary antibody. Indirect methods provide higher signal intensity because several labeled secondary antibodies can bind indirectly to each antigen (Fig. 17). Indirect detection may also include amplification steps to increase signal intensity.

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.

Table 2. Comparison of direct vs indirect detection methods.

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. 18, 19, and 20). 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.

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.

Labels, such as fluorescent dyes, proteins, enzymes, and biotin, are conjugated to secondary antibodies to visualize the target protein's presence.

·        Fluorescent labels emit light in the visual range when excited by light of a wavelength. There are several available, all with their own excitation and emission characteristics.

·        Enzymatic labels, such as horseradish peroxidase (HRP) and alkaline phosphatase (AP), 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), which is avidin or streptavidin conjugated to an enzyme or fluorochrome.

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 3).

Table 3. Choosing a secondary antibody labeled with an enzyme or fluorochrome for different applications.

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 on 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.

• Select the brightest fluorophore to label a protein with the lowest expression levels

• All secondary antibodies should come from the same host species when you use multiple labels

• Pre-adsorbed secondary antibodies are helpful for multicolor analysis to ensure low cross-species reactivity

• Fragment antibodies are smaller and penetrate tissues more efficiently (useful for IHC)

• Biotin conjugates can detect low-abundance proteins

Here we discuss all essential factors you need to consider to successfully validate antibody specificity in your experimental setup, such as choosing and preparing the appropriate positive and negative controls and validating antibodies in specific applications.

Antibody validation revolves around proving three key aspects:

·        Specificity and functionality - showing an antibody can differentiate between various antigens in the intended application.

·        Affinity - showing the strength of binding between antibody and epitope.

·        Reproducibility - showing that your validation data can be reproduced in any lab.

Here we will focus on how you can validate antibody specificity in your experimental setup to ensure accurate and consistent results. Although a good manufacturer usually tests an antibody in several applications and species, it's impossible to account for numerous protocols and reagents with which researchers may use the antibody. Therefore, your antibody validation steps are essential because they are specific to your setup.

1. Choice and preparation of positive and negative controls

Identifying and using appropriate positive and negative controls is essential for successful antibody validation.

·        A positive control is a relevant cell line or tissue sample strongly expressing the target protein of interest that can be used to confirm the selective binding of your antibody.

·        A negative control is a cell line or tissue sample that does not express the target protein and, therefore, can provide the data on the non-selective binding properties of your antibody. When a true negative control is not available, a sample expressing low levels of the target proteins can work as an acceptable alternative.

It's often challenging to determine cell or tissue types that do or do not express the target protein. You can find information about target protein expression in different tissues or cell lines in peer-reviewed papers and online protein databases, including:

• Proteomics DB

• GeneCards

• Cancer Cell Line Encylopedia

• Gene (NIH)

• Protein Atlas

• UniProt

However, there are a few limitations to defining protein expression profiles using online databases:

• Expression data may not be complete in some cases

• It can be challenging to find negative cell lines or tissues, particularly for essential housekeeping genes. 

• RNA levels are often very unreliable in determining the quantity of proteins.

Furthermore, antibodies may or may not recognize the protein in its native or denatured state. It is therefore essential to prepare the test samples accordingly. For example, an antibody that recognizes the protein only in its native form should not be used on samples using denaturing conditions, such as western blot.

2. Protocols

Ensure you use an optimized protocol to give the antibody the best chance of passing the validation process. For instance, incubation times can vary dramatically from a minimum of one hour to overnight at 4°C, so you'll need to determine the optimal incubation period for each antibody. If the incubation period is too short, you may encounter sensitivity issues, while prolonged incubation time may lead to background staining. You will also need to optimize other factors, such as working dilutions, blocking conditions, and the use of native vs denatured conditions.

3. The choice of buffers

The majority of antibody assays will use two buffer types: PBS or TBS. You will need to determine the optimal buffer for your experiment, considering parameters that can influence buffer performance, such as pH.

Models for designing positive and negative controls

Cell lines or tissues that endogenously express or lack the target protein can serve as positive or negative controls, respectively. You can use several various cell lines with different protein expression levels to provide a range of controls. Alternatively, appropriate positive and negative controls can be designed using multiple methods, including knock-out models, siRNA knockdown and cell treatment (Table 4).

Table 4. Models for designing appropriate positive and negative controls.

Validating antibody specificity in various applications

You can use several different methods to validate antibodies. Below we outline some popular applications, their benefits, and limitations.

Note that the two following methods can't be considered exhaustive tests of antibody specificity; therefore, we do not recommend using them alone:

·        Blocking with an immunizing peptide can confirm that an antibody binds its immunogen. However, the immunizing peptide will block both specific and non-specific antibodies, so it can't serve as a comprehensive method to confirm antibody specificity.

·        Omitting a primary antibody can evaluate the tissue or secondary detection reagents but not the primary antibody specificity.

With proper storage and handling, most antibodies should retain activity for months, if not years. In this section, we provide general guidelines on antibody storage and handling. Please always refer to the manufacturer's datasheet for specific storage recommendations.

Upon receiving the antibody, you will need to centrifuge it at 10,000 x g for 20 seconds to pull down the solution trapped in the vial threads and then aliquot it into low-protein-binding microcentrifuge tubes. Aliquotting minimizes damage due to repeated freeze/thaw cycles that can denature an antibody, causing it to form aggregates that reduce its binding capacity. Aliquotting also helps minimize contamination introduced by pipetting from a single vial multiple times.

Aliquots should be frozen and thawed once, with any remainder kept at 4°C. It's usually recommended to store antibodies at -20°C as there's no significant advantage to storing them at -80°C.

The size of the aliquots will depend on how much you typically use in an experiment. Aliquots should be no smaller than 10 μL. The smaller the aliquot, the more the stock concentration is affected by evaporation and adsorption of the antibody onto the surface of the storage vial.

In most cases, storage at 4°C upon receiving the antibody is acceptable for one to two weeks. It is essential to follow the recommendations on the datasheet.

Make sure not to use a frost-free freezer: it's unlikely your lab would, but the cycling between freezing and thawing should be avoided. For the same reason, antibody vials should be placed in the freezer area with minimal temperature fluctuations, for instance, towards the back rather than on a door shelf. 

Some researchers add the cryoprotectant glycerol to a final concentration of 50% to prevent freeze/thaw damage because glycerol lowers the freezing point to below -20°C. While this may be acceptable for many antibodies, you should check the datasheet to see if the manufacturer tested antibody stability in this storage condition.  

Storing solutions with glycerol at -80°C is not advised since this is below the freezing point of glycerol. Also, glycerol or any other cryoprotectant can be contaminated with bacteria, so you must obtain a sterile preparation.

Conjugated antibodies often require additional storage and handling precautions since they're more complicated than non-conjugated antibodies. For example, conjugated antibodies – whether conjugated to fluorochromes, enzymes, or biotin –  should be stored in dark vials or wrapped in foil because exposure to light will compromise conjugates' activity. Fluorescent conjugates, in particular, are susceptible to photo-bleaching and should be protected from light during all phases of an experiment.

Table 6 below provides the detailed guidelines for proper conjugated antibody storage and handling.

Table 6. Handling, aliquoting, and storage guidelines for conjugated antibodies.

*Freezing and thawing enzyme-conjugated antibodies will reduce enzymatic activity and affect the antibody binding capacity. Therefore, enzyme-conjugated antibodies should not be frozen at all and should instead be kept at 4°C unless an antibody contains a cryoprotectant, and its stability has been validated for long-term storage at -20°C.

To prevent microbial contamination, you can add sodium azide to an antibody solution to a final concentration of 0.02% (w/v). If an antibody already contains this preservative, this will be indicated on the datasheet in the storage buffer section.

Sodium azide should be avoided when staining or treating live cells with antibodies or conducting in vivo studies. This antimicrobial agent is toxic to most other organisms as it blocks the cytochrome electron transport system.

Sodium azide will interfere with any conjugation involving an amine group and should be removed before proceeding with the conjugation. After conjugation, you can store antibodies in sodium azide, except for HRP-conjugated antibodies, since sodium azide inhibits HRP.  An acceptable alternative to sodium azide is 0.01% thimerosal (merthiolate), which does not have a primary amine. Also, sodium azide can be removed from antibody solutions by dialysis, ultrafiltration or gel filtration.

Proteins A and Protein G (expressed by Staphylococcus aureus or Streptococcus bacteria, respectively) are antibody binding proteins often used in antibody purification. Protein A/G purification is based on protein A or G's high affinity to the immunoglobulin Fc domain. Protein A/G purification eliminates the bulk of the serum proteins from the raw antiserum. However, it does not eliminate the non-specific immunoglobulin fraction. As a result, the protein A/G purified antiserum may still possess some undesirable cross-reactivity.

If an antibody is received in an unpurified format, you may need to purify it before using it in your experimental setup. Antibody purification methods range from very crude to highly specific, and the necessary level of the purification depends on your intended application for the antibody. Here we briefly overview the most common unpurified antibody formats and antibody purification methods.

Polyclonal antibodies are often available in relatively unpurified forms, such as "serum" or "antiserum". Antiserum refers to the blood serum from an immunized host containing antibodies of interest (as well as other serum proteins and antibodies).

In addition to antibodies recognizing the target antigen, antiserum contains antibodies to various non-target antigens that sometimes react non-specifically in immunological assays. For this reason, raw antiserum is often purified to eliminate serum proteins and enrich the immunoglobulin fraction that specifically reacts with the target antigen.

Monoclonal antibodies can be produced using hybridoma cell cultures (cytokine-secreting cells) and harvested as hybridoma tissue culture supernatants. Please refer to Chapter 2 for more information about monoclonal antibody production.

Ascites fluid is a historical in vivo antibody production method, which is now only used in exceptional cases, ie, when an antibody can't be produced by in vitro technologies.

In this method, monoclonal antibodies are produced by growing hybridoma cells within the peritoneal cavity of a mouse (or a rat). The hybridoma cells are injected into a host's abdomen, where they multiply and generate fluid (ascites), which can be harvested. This ascites fluid contains a high antibody concentration, usually providing higher antibody yields than hybridoma cell culture. However, the ascites fluid also includes many non-specific immunoglobulins from the host.

Antibody purification is achieved by selective enrichment or specific extraction of antibodies from serum (for polyclonal antibodies), ascites fluid, or cell culture supernatant of a hybridoma cell line (for monoclonal antibodies). Below we describe typical purification methods for polyclonal antiserum or monoclonal ascites fluid/tissue culture supernatant.

Proteins A and Protein G (expressed by Staphylococcus aureus or Streptococcus bacteria, respectively) are antibody binding proteins often used in antibody purification. Protein A/G purification is based on protein A or G's high affinity to the immunoglobulin Fc domain. Protein A/G purification eliminates the bulk of the serum proteins from the raw antiserum. However, it does not eliminate the non-specific immunoglobulin fraction. As a result, the protein A/G purified antiserum may still possess some undesirable cross-reactivity.

Affinity purification isolates a specific protein or group of proteins with similar characteristics using affinity tags. The technique separates proteins based on a reversible interaction between the protein and a specific ligand coupled to a chromatographic matrix (Fig. 19).

Antigen affinity purification takes advantage of the specific immunoglobulin fraction's affinity for the immunizing antigen against which it was generated. This purification method eliminates the bulk of the non-specific immunoglobulin fraction while enriching the immunoglobulin fraction that specifically reacts with the target antigen. The resulting affinity-purified immunoglobulin will primarily contain the immunoglobulin of the desired specificity.

Polyclonal antibodies are sometimes pre-adsorbed, meaning they have been adsorbed with other proteins, or serum from various species, to eliminate any antibodies that may cross-react. The solution containing secondary antibodies is passed through a column matrix containing immobilized serum proteins from potentially cross-reactive species (Fig. 20). Non-specific secondary antibodies are retained in the column, while highly specific secondaries flow through. The resulting purified antibody should exhibit significantly reduced cross-reactivity.

When using an antibody for the first time, you may need to optimize its dilution for your specific application and experimental setup. Here we describe how to define the optimal antibody concentration by titration and provide suggested dilutions for antibodies without recommended dilution on the datasheet.

The rate of binding between antibody and antigen – affinity constant – can be affected by temperature, pH, and buffer constituents. Varying the relative concentrations of an antibody and an antigen solution can also control the extent of antibody-antigen complex formation. As it is not usually possible to change the antigen concentration, the optimal working concentration of each antibody must be determined with dilutions for each application and set of experimental conditions.

Usually, antibodies have recommended dilutions for various applications included in the datasheet. However, they may require some optimization in your specific experimental setup.

The optimal antibody concentration, which gives the best staining with minimum background, must be determined experimentally for each assay and is usually determined using a series of dilutions in a titration experiment. For example, if a product datasheet suggests using a 1:200 dilution, it is recommended to make dilutions of 1:50, 1:100, 1:200, 1:400 and 1:500.

A titration experiment is done by first selecting a fixed incubation time and then a series of experimental dilutions of the antibody. Each dilution should be tested on the same sample type to keep the same experimental conditions.

Many antibodies will have similar batch-to-batch consistency; therefore, only one titration experiment is required in most cases. However, especially for polyclonal antibodies, when there is a change in the staining results between batches of the same antibody, we recommend performing another titration experiment.

Unpurified antibody preparations differ significantly in antibody concentration. If the specific antibody concentration of a given unpurified antibody preparation is unknown, we recommend using a concentration/purification kit and refer to the table below as a guideline.

Table 7 provides various dilutions for each application for different antibody formats.

Table 7. Suggested antibody dilutions for different applications.

EIA=enzyme immunoassays, FACS=fluorescence-activated cell sorting, ICC=immunocytochemistry, IHC=immunohistochemistry, IP=immunoprecipitation, WB=western blot.