Antibodies for Western Blotting | Thermo Fisher Scientific

Invitrogen validated western blot antibodies

The right antibodies are essential for clean, definitive, and reproducible western blot results. Use the tabs below to find information on aspects to consider when selecting primary and secondary antibodies for western blot experiments.

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Primary antibody selection considerations for western blot analysis

Polyclonal vs monoclonal vs recombinant antibodies

	Polyclonal	Monoclonal	Recombinant
Definition	Collection of antibodies from different B cells that recognized multiple epitopes on the same antigen	Single antibody produced by identical B cell clones that recognize one epitope on the same antigen	Single antibody derived from recombinant DNA. Can be modified on the DNA level or used to generate defined antibody pool.
Advantages	Highly sensitive. Many antibodies in the polyclonal pool can bind epitopes on antibody target	Lot-to-lot consistency. Often well characterized, historic knowledge of specific clones, publications for performance in western blotting.	Stable, long-term supply with lot-to-lot consistency. Not susceptible to cell-line drift.
Disadvantages	Lot-to-lot variability of antibody pool can result in inconsistent detection. Epitopes similar to target can contribute to detection of unspecific bands.	Sensitivity of detection is dependent on abundance and exposure of a single epitope. Cell line drift could result in subtle, long-term changes to antibody.	Very specialized and epitope dependent. Longer development time. May need more upfront optimization. Usually higher price.

Polyclonal, monoclonal and recombinant antibodies all work well for western blotting. Polyclonal antibodies are a pool of many monoclonal antibodies, which can vary from immunization to immunization and lot-to-lot. Polyclonal antibodies recognize multiple epitopes of an antigen and are therefore usually more sensitive than monoclonal antibodies that recognize only one epitope. This can be a benefit when epitope abundance, epitope masking or epitope exposure is a concern. Polyclonal antibodies are less expensive and less time-consuming to produce. Monoclonal antibodies are valued for their lot-to-lot consistency and in many cases, extensive characterization and publication history. Monoclonal antibodies are usually produced by cell lines that generate one individual antibody clone. These cell lines (or hybridomas) are grown in cell culture when the antibody is needed for production. Just like any often-propagated cell line, these cell lines could potentially undergo gradual changes affecting antibody production yields or even antibody characteristics. Recombinant antibodies are the best option for consistent, in vitro-derived antibody production and lot-to-lot consistency. Recombinant antibodies are produced by transfecting production cell lines with recombinant DNA that encodes the desired immunoglobulins. Recombinant antibodies have several benefits: They can be modified at specific sites to add desired characteristics to IgGs and they are not subject to cell line drift such as hybridoma derived monoclonals. Recombinant antibodies can be pooled to generate recombinant antibody pools, such as recombinant polyclonal primary antibodies or superclonal recombinant secondary antibodies.

Antibodies are usually provided purified in PBS or similar buffers; however in some cases, crude antibody preparations such as serum or ascites fluid are necessary in order to maintain certain antibody characteristics or antibody yield. It is important to optimize western blotting protocols to minimize the impact of impurities present in crude antibody preparations on background.

Direct vs indirect detection

Both direct and indirect methods of detection can be used in western blotting. Each method provides its own advantages and disadvantages. With the direct detection method, an enzyme- or fluorophore-conjugated primary antibody is used to detect the antigen of interest on the blot. In the indirect detection method, an unlabeled primary antibody is first used to bind to the antigen. Subsequently, the primary antibody is detected using an enzyme- or fluorophore-conjugated secondary antibody. The indirect method offers many advantages over the direct method, which are described below.

	Direct method	Indirect method
Description	Primary antibody is conjugated with an enzyme or fluorescent dye for direct detection.	Unlabeled primary antibody is detected using an enzyme- or fluorophore-conjugated secondary antibody
Advantages	Requires only one antibody Saves time by eliminating secondary antibody incubation step Eliminates possible background by secondary antibody cross-reactivity in certain samples.	Signal amplification, since multiple labeled secondaries bind to each primary Many options using HRP, Alexa Fluor or Alexa Fluor Plus labeled secondary antibodies Save time with multiplexed detection using fluorescent secondary antibodies Further signal amplification possible with biotinylated secondaries and fluorescent or enzyme-labeled streptavidin.
Disadvantages	Label may interfere with target binding, resulting in lower sensitivity and higher background. Selection of directly conjugated primary antibodies is limited	Additional steps required Potential for non-specific binding that may increase background in certain samples.

Host species considerations

Multiple species are used to generate antibodies that can be used in western blot applications. Most commonly: mouse, rabbit, rat, goat, donkey and chicken. Which host species primary antibody to choose will depend on whether a single target is being probed or multiple targets are being probed in a multiplex western blot experiment. When investigating only one antigen at a time, theoretically any host species can be used, however most primary research antibodies for western blotting are produced from immunized rabbits (polyclonal, monoclonal, recombinant) or mice (hybridoma derived monoclonals). Some host species provide additional advantages over others for example due to their size or immune biology. When comparing mouse or rabbit for example, rabbits usually are better at tolerating immunizations and have a significantly longer life span than mice. Furthermore, rabbits exhibit a more diverse natural repertoire of antibodies than mice, which makes rabbits a popular host for the generation of polyclonal, monoclonal and rabbit recombinant antibodies. When aiming to generate the most suitable monoclonal or recombinant antibody for western blotting, the greater repertoire of rabbit-produced antibodies allows for more successful screening, isolation and cloning of high affinity recombinant antibodies. This is especially important when aiming to make western blot antibodies to more challenging epitopes that may not be feasible to produce with other systems.

When performing a multiplex western blot, use primary antibodies from different host species for each target being probed. Ideally, use a combination of antibodies from two distantly related species such as rat and rabbit, avoiding combinations like mouse and rat or goat and sheep. This will aid in the selection of appropriate secondary antibodies to minimize potential antibody cross-reactivity, which can lead to confusing results.

Multiplexing host considerations

First antibody host species	Secondary antibody host species
Rat	Rabbit	✓
Mouse	Rabbit	✓
Goat	Rabbit or mouse	✓
Mouse	Rat	X
Goat	Sheep	X

Western blot validated antibodies

Although antibodies are designed to recognize a specific target antigen, they may not work equally in all applications. Choose antibodies designated specifically for western blotting or that list western blotting as an application. In addition, it is important to confirm that the antibody is specific towards the native or denatured protein, to determine if SDS-PAGE or native PAGE should be performed.

When selecting a primary antibody, confirm:

Antibody is validated for western blotting
Antibody specificity towards the native or denatured protein

Invitrogen antibodies undergo a rigorous 2-part testing approach. Part 1: Target specificity verification, Part 2: Functional application validation. Target specificity verification helps ensure the antibody will bind to the correct target. Most antibodies were developed with specific applications in mind. Testing that an antibody generates acceptable results in a specific application is the second part of confirming antibody performance. Learn more about Invitrogen Antibody Validation process.

Secondary antibody selection guide for western blot analysis

Target species (the primary antibody)

When choosing secondary antibodies for western blotting, one of the main selection criteria is the species of the primary antibody to which the secondary antibody binds. For example, if the primary antibody is a rabbit polyclonal IgG, then an anti-rabbit IgG secondary antibody raised in another host species, for example goat, donkey or mouse can be used to detect the primary antibody (e.g., a goat anti-rabbit IgG secondary antibody).

For the detection of multiple specific protein targets by western blot, it can be beneficial to use primary antibodies from different species. Matching secondary antibodies that are labeled with different fluorescent dyes (for example Alexa Fluor Plus 488, 555, 647, 680 or 800), can then be used to perform multiplex fluorescent western blotting.

We offer a wide range of labeled secondary antibodies that are suitable for fluorescent or chemiluminescent western blotting. Find your new secondary to match your primary antibody with over 16 target species to choose from.

Secondary target specificity and implications for secondary antibody selection

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Secondary antibodies can be generated to bind to whole primary antibody immunoglobulins, for example H+L (heavy and light chains) targeting secondary antibodies, or they can be generated to only bind specific parts of the Immunoglobulin, for example kappa vs. lambda light chain specific secondary antibodies, heavy vs. light chain specific secondaries, or fragment specific secondary antibodies. Examples are Fc fragment specific secondaries, F(ab) or F(ab’)2 specific secondary antibodies (see figure).

When planning western blot experiments, secondary antibody selection in addition to other factors, such as optimizing antibody dilution, can be instrumental in obtaining optimal results. Thoughtful selection of the secondary antibody and dilution optimization can significantly improve western blot analysis, especially when dealing with varying protein target abundance on the same western blot membrane, or high affinity vs. lower affinity primary antibodies. Varying target abundance is very common, since loading control housekeeping proteins are very abundant in lysates, while many protein targets of interest may be expressed only at low copy numbers. Visualizing both on the same membrane can be challenging. The table below provides general guidelines that can help improve western blotting detection by using the unique features of different secondary antibodies.

Secondary antibody target	Advantages	Disadvantages
H+L chain specific: multiple secondary antibodies bind to heavy chain of primary antibody and light chains on all immunoglobulins	Most versatile choice. Antibodies bind to heavy and light chains, regardless of primary antibody structure Recommended for high signal amplification of protein targets in chemiluminescent and fluorescent western blots Obtain high sensitivity and low background with cross-adsorbed or highly cross-adsorbed antibodies.	Highly abundant target detection may result in signal saturation outside of linear detection range at high antibody concentrations. Cross-adsorbed or highly cross-adsorbed H+L secondaries are recommended to minimize cross reactivity with IgG binding proteins in sample. Possible cross-reactivity with light chains of other immunoglobulins. For example all goat anti-rabbit IgG (H+L) secondaries will not only bind to rabbit IgG heavy chains (gamma chains), but also to light chains from all rabbit immunoglobulins, such as IgM, IgE or IgA.
Fc fragment specific: secondary antibodies bind to Fc portion of heavy chain	Good choice for the detection of mouse monoclonal primary antibodies. Binding is independent of light chain specificity. Can be used after immunoprecipitation to bind native primary antibody IgG in cases where the primary antibody target runs at ~25kD. The Fc specific secondary only detects the 50kD heavy chain of the denatured IgG and does not bind the 25kD light chain. Can be used to detect specific immunoglobulin isotypes and subclasses that differ in the Fc part of their heavy chains. This property enables the multiplex detection in fluorescent WB by using primary antibodies of different isotypes and subclasses. Fc fragment specific secondary antibodies are versatile as they can also be used as capture or detection antibodies in immunoassays.	Usually less sensitive than H+L specific secondary antibodies. Possible interference from Fc receptors in immune cell samples. When used after immunoprecipitation, the Fc specific secondary antibody binds native primary antibody IgG as well as the 50kD heavy chain of denatured IgG. This can interfere with target detection if the primary antibody protein target runs at ~50kD.
F(ab) or F(ab’)2 specific secondary antibodies bind to F(ab) or F(ab’)2 portion of primary antibody, detecting heavy or light chains	F(ab) and F(ab’)2 specific secondary antibodies are not commonly used in western blotting since most primary antibodies for western blotting consist of both heavy and light chains. The primary antibody Fc region serves the purpose of allowing for additional space on the immunoglobulins for secondary antibody binding, enabling more sensitive target detection.
Light chain specific	Binding is independent of heavy chain specificity. Secondary antibodies will bind to all immunoglobulin classes and isotypes that share the same light chains. Kappa vs. Lambda chain specific secondary antibodies can be used for additional specificity when detecting a primary antibody with known light chain composition. Can be used after immunoprecipitation to bind native primary antibody IgG in cases where the primary antibody target runs at ~50kD. The light chain specific secondary only detects the 25kD light chain of the denatured IgG and does not bind the 50 kD heavy chain.	Usually less sensitive than H+L specific secondary antibodies. When used after immunoprecipitation, the light chain specific secondary antibody binds native primary antibody IgG as well as the 25 kD light chain of denatured IgG. This can interfere with target detection if the primary antibody protein target runs at ~25kD.

Host species and secondary antibody types (polyclonal, monoclonal, recombinant)

Polyclonal, monoclonal and recombinant secondary antibodies, as well as antibody fragments can be used for western blotting. Polyclonal secondary antibodies are the most common form of secondary antibodies in use. They recognize multiple epitopes on a primary antibody and are therefore more sensitive than monoclonal antibodies that recognize only one epitope or antibody fragments without an Fc region. Monoclonal secondary antibodies are valued for their lot-to-lot consistency and in many cases, extensive characterization and publication history. Recombinant secondary antibodies have additional benefits. In addition to being well characterized and showing lot-to-lot consistency, they can be further modified at specific sites to add desired characteristics to the native immunoglobulin, such as class switching or site specific labeling. Recombinant antibodies can be pooled to generate recombinant antibody pools, such as recombinant polyclonal primary antibodies or superclonal recombinant secondary antibodies. The table below shows advantages and disadvantages of polyclonal, monoclonal and recombinant antibodies as they apply to secondary antibodies.

	Polyclonal	Monoclonal	Recombinant
Definition	Collection of secondary antibodies from different B cells that recognized multiple epitopes on the same immunoglobulin. Can be specific to any part of an immunoglobulin, such as Fc or F(ab). Most commonly used to generate secondaries that detect H+L chains.	Single antibody produced by identical B cell clones that recognize one epitope on the same immunoglobulin antigen. They can be specific to kappa, lambda light chains, as well as isotype and subclass.	Single antibody derived from recombinant DNA. Can be modified on the DNA level or used to generate defined antibody pool.
Advantages for WB	Highly sensitive. Many antibodies in the polyclonal pool can bind epitopes on primary antibody.	Lot-to-lot consistency. Often well characterized, historic knowledge of specific clones, publications for performance in WB.	Stable, long-term supply, lot-to-lot consistency. Can be modified for desired characteristics for improved performance.
Disadvantages for WB	Lot-to-lot variability is possible, but usually less critical than for primary antibodies.	Sensitivity of detection is dependent on abundance and exposure of a single epitope. Cell line drift could result in subtle, long-term changes to antibody.	Very specialized and epitope dependent. Longer development time. May need more upfront optimization. Usually higher price.

Detection method

Secondary antibodies can be conjugated to several different probes or enzymes for detection of the target antigen. The choice of label depends upon the application and how the secondary antibody is going to be detected. Enzyme reporters such as horseradish peroxidase (HRP) and alkaline phosphatase (AP) are the most commonly used in western blotting. These enzymes can be used with either chemiluminescent or chromogenic detection methods. Fluorescent labeled secondary antibodies are also becoming more widely used with increasing signal to noise ratios for detecting low abundant targets.

Compare the different western blotting detection methods

Cross-adsorbed antibodies

When performing multiplex western blot analysis, consider using secondary antibodies that are highly cross-adsorbed to limit cross reactivity between antibodies. Cross-adsorption is a purification process that helps eliminate nonspecific antibodies in an antibody mixture, such as antibodies of specific classes, isotypes, or host species.

Read more about cross-adsorption and cross reactivity

WB analysis after immunoprecipitation

Secondary antibody selection can play an important role when performing western blotting after immunoprecipitation. When performing an immunoprecipitation, the primary antibody or control IgG, are usually co-eluted with the target protein. The elution fraction that is used for western blot analysis therefore always contains variable amounts of IgG. For example, if a rabbit polyclonal antibody was used to enrich a target protein by immunoprecipitation, the reduced and denatured heavy and light chains in the elution fraction would produce bands at 25 and 50kD when using a traditional HRP conjugated anti-rabbit IgG (H+L) secondary antibody due to the secondary antibody binding directly to the denatured IgG.

If heavy and light chains produced by the secondary antibody don’t interfere with the primary antibody target of interest, one merely has to mark these bands as heavy and light chains. However, in cases when the target of interest may run at around ~25 or 50kD, it may not be possible to distinguish the target band from the heavy or light chain bands.

There are several solutions that can be applied:

Use Clean-Blot IP Detection Reagent, which only detects the native primary antibody and not the denatured IgG from the immunoprecipitation elution fraction.
Use an Fc specific secondary antibody if the target of interest is ~25kD (light chain interference)
Use a light chain specific secondary antibody, if the target of interest is ~50kD (heavy chain interference)

See other factors to consider when picking secondary antibodies

Finding the right loading controls

Choosing a loading control or housekeeping protein is an important aspect of western blot normalization. For various reasons not all loading controls can be equally utilized for normalization studies in all biological test systems. For example, if a chemiluminescence or one-color fluorescence system is being used for target detection, the housekeeping protein should not interfere with detection of the target (e.g., should not be of similar molecular weight). In addition, the quantitative accuracy and linear range of the loading control within the biology test system should be assessed before using for western blot normalization. The signal obtained for loading control should be linear over a wide concentration range, such that it can be used as a reliable reference for normalization.

Continue reading on how to normalize western blots using loading controls

Loading control antibodies are available in unconjugated form, or conjugated form with biotin, HRP, or fluorescent dyes.

Type of loading control	Unconjugated	Biotin	HRP	Alexa Fluor
Beta-actin antibodies	MA5-15739 MA1-140 MA1-91399 PA1-46296	MA5-15739-BTIN	MA5-15739-HRP	MA1-140-A488 MA1-140-A555 MA1-140-A647
Beta-tubulin antibodies (BT7R)	MA5-16308 MA1-118 MA1-19164 PA1-41331 PA1-16947	MA5-16308-BTIN	MA5-16308-HRP	MA5-16308-A488 MA5-16308-A555 MA5-16308-A647
Cyclophilin D antibodies	PA3-022 PA1-028 PA3-023
GAPDH antibodies	MA5-15738 39-8600 PA1-987 PA1-988	MA5-15738-BTIN	MA5-15738-HRP	MA5-15738-A488 MA5-15738-A555 MA5-15738-A647

Application notes and technical guides

Primary antibodies