What is western blot?

Western blot, or western blotting, is a technique widely used in research to separate and identify specific proteins within a complex mixture. Western blot allows us to determine the relative protein levels between samples and establish the molecular weight of the target, which can provide insight into its post-translational processing. To achieve this, western blot implements three steps: (1) separation by size, (2) transfer to a solid support, and (3) visualizing target protein using primary and secondary antibodies.

In the first step, the proteins are separated based on size by gel electrophoresis. The gel is then placed in contact with the membrane, and the use of an electrical current induces the proteins to migrate from the gel to the membrane. Protein transfer to the membrane is essential because gels used for electrophoresis provide a poor surface for immunostaining, ie, antibodies don’t stick to the proteins in the gel. The membrane can be further processed with antibodies specific to the target of interest and visualized using enzyme-linked or fluorophore-conjugated secondary antibodies and detection reagents.

Choosing antibodies for western blot

The sensitivity and specificity of your western blot depend on the quality of the antibodies and the experimental conditions they are used in. When working with tissue lysates or tissue culture supernatants containing serum and, therefore, endogenous immunoglobulins, you should select a primary antibody raised in species different from that of your sample. For example, if you are studying a mouse protein, choose a primary antibody raised in a species other than mouse (eg, primary antibody raised in rabbit). This is to avoid cross-reactivity of the secondary anti-immunoglobulin antibody with endogenous immunoglobulins in the sample. The choice of host species of the primary antibody is less critical when using samples that don’t contain endogenous immunoglobulin.

Whenever possible, choose a primary antibody that has been knockout (KO) validated to ensure it binds specifically to the intended target.

Choosing conjugated secondary antibodies

To visualize your protein, select a secondary antibody (against the host species of the primary antibody) that will bind to the primary antibody. Using an enzyme-linked secondary antibody, such as horseradish peroxidase (HRP)- or alkaline phosphatase (AP)-conjugate antibody, or a western blot-optimized fluorescence conjugate, offers a high level of sensitivity. Remember to check that the light emission wavelength of a conjugate is compatible with your reading device/scanner.

The increased sensitivity of conjugated secondary antibodies, compared to primary antibody only, results from these antibodies binding to the primary antibody at multiple locations, which amplifies the signal. Therefore, using secondary antibodies is ideal for western blot since their signal amplification allows for easier detection of the protein of interest in a complex protein mixture.

Choosing directly conjugated primary antibodies

Using directly conjugated primary antibodies (eg HRP-conjugates) in western blot allows you to speed up and simplify the protocol, omitting the need for the secondary antibody step. 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 HRP suitable for western blot. If your antibody of choice is not available in a suitable conjugated format, you can use abcam’s antibody conjugation kits.

Direct and indirect labeling for western blot

Before running a western blot, it is extremely important to research the target protein thoroughly. To learn more about the procedure, refer to our western blot protocol

1. In a traditional western blot (indirect labeling), protein samples are first resolved by SDS PAGE and then electrophoretically transferred to the membrane.
2. After a blocking step, the membrane is probed with a primary antibody that was raised against the antigen in question.
3. Following a washing step, the membrane is typically incubated with an enzyme-conjugated secondary antibody directed against the primary antibody. In the case of fluorescent detection, a fluorophore-conjugated secondary antibody is used instead.
   • The fluorescence of the dye or activity of the enzyme, such as alkaline phosphatase (AP), glucose oxidase (GO), or horseradish peroxidase (HRP), is necessary for signal generation.
4. Finally, the membrane is washed again and incubated with an appropriate enzyme substrate (if necessary), producing a reportable signal.
   • Direct labeling analysis uses conjugated primary antibodies (eg, HRP-conjugated), which eliminates the need for the secondary antibody step, thereby simplifying the procedure, shortening the protocol, and expediting the time to results.

Immunoblot detection

There are several different choices of readout when western blotting. Each has advantages and disadvantages, which depend on your needs and the equipment available in your lab.

Colorimetric western blotting

Figure 1. Schematic representation of colorimetric western blot detection. The left panel demonstrates indirect detection, while the right panel shows direct detection.

Colorimetric detection relies on generating a colored product that becomes deposited on the western blot, which is formed following the conversion of a chromogenic blotting substrate by an appropriate enzyme. The limited sensitivity of chromogenic substrates can make it difficult to optimize them for detecting proteins of low abundance. However, the chromogenic reaction can be allowed to develop for several hours (or even overnight) to allow the background signal to develop simultaneously.

In contrast, colorimetric substrates are perfect for detecting abundant proteins since the reaction can be monitored visually and allowed to progress until there is adequate color development before being stopped. No specialized equipment is required to visualize the colored precipitate, and the produced signal is highly stable.

Fluorometric western blotting

Figure 2. Schematic representation of fluorescent western blot detection.​​

Fluorometric detection requires the use of an antibody, which has been labeled with a fluorophore. A light source is used to excite the fluorophore, producing a transient light emission as it returns to its ground state. The light is emitted at a higher wavelength than that used for excitation and is detected with a specialized reader, such as a fluorimeter scan.

Chemiluminescent western blotting

Figure 3. Schematic representation of chemiluminescent western blot detection.

Chemiluminescence occurs when an enzyme catalyzes a substrate and produces light as a by-product of the reaction. The limiting reagent in the reaction is the substrate – as this is exhausted, the light production decreases and eventually stops. However, a well-optimized procedure should produce a stable light output for several hours, allowing consistent and sensitive protein detection.

To visualize the light signal, you can use a developer machine if working with X-ray films or a western blot camera device, which requires no X-ray films.

For more information, check out our library of western blot resources.