Western Blotting


What is Western blotting and how does it work?

Western blotting is a widely used analytical technique that can identify one or more specific proteins in a complex mixture of proteins.

What does Western blotting detect?

Western blotting is able to detect specific protein molecules from a mixture of proteins. It is a powerful tool that provides information about the presence, size, and under the right conditions, even the amount of a protein. 

The basic steps of Western blotting

Though commonly used and often routine in many labs, Western blotting can be source of frustration when it doesn’t work. The key to the best Westerns is understanding the process. Below is a brief overview of each step.

A Western blotting experiment typically involves these five steps:

  1. Gel electrophoresis
  2. Transfer of the proteins to a solid membrane
  3. Blocking the membrane to prevent nonspecific binding of antibodies
  4. Probing the membrane with an antibody
  5. Detecting the antibody using colorimetric, radioactive, chemiluminescent, or fluorescent detection methods

1. Gel electrophoresis

Gel electrophoresis is the first step of a Western blot, where proteins are physically separated from one another across a gel matrix in a process called gel electrophoresis (Figure 1.1).

The protein sample is mixed with a loading buffer, loaded onto the gel, and then subjected to an electrical current. The proteins are negatively charged under the experimental conditions. They then travel through the gel towards the positive electrode.

Polyacrylamide gel electrophoresis process
Figure 1.1. Polyacrylamide gel electrophoresis

Depending on the type of gel and buffer system used, the distance a protein migrates through the gel matrix is governed primarily by the mass:charge ratio of the individual protein or simply the molecular weight of the protein.

Protein electrophoresis can be run under a variety of buffer systems and gel compositions that change the relative migration of proteins.

2. Transfer to membrane

After electrophoretic separation of proteins through the gel, the proteins are transferred to a solid membrane support for subsequent steps. Efficient transfer relies on the choice of membrane, the type of transfer apparatus used, and the composition of the transfer buffer. Successful transfer of proteins relies on both the migration of proteins out of the gel and retention of proteins on the membrane. Like gel electrophoresis, the transfer step uses electricity to move negatively charged proteins towards the positively charged electrode (Figure 1.2).

Semi-dry transfer stack for electrophoresis
Figure 1.2. A typical semi-dry transfer setup

Membranes commonly used for Western blotting include nitrocellulose (NC) and polyvinylidene difluoride (PVDF). Buffer components are optimized based on the type of transfer system being used (wet or semi-dry), the type of gel employed, the choice of membrane, and the protein of interest. Transfer times and voltage settings should be optimized for each transfer.

While proteins generally transfer more rapidly at higher voltages, transfer efficiency is not always consistent. Insufficient current and/or time may result in incomplete transfer, while high current and/or lengthy transfer times may result in loss of proteins via transfer through the membrane without retention.

3. Membrane blocking

To keep background signal as low as possible, the membrane is incubated in a blocking solution after transfer to prevent nonspecific binding of antibodies. Optimizing blocking conditions is important for obtaining high-quality data, especially when quantitative information is desired.

There are several different types of blocking agents are available, and the blocking solution should be optimized for each antibody:antigen interaction.

4. Membrane incubation with antibody

After blocking, the membrane is probed with antibody and then unbound antibody washed away. There are two types of detection: direct and indirect (Figure 1.3)

For direct detection, the primary antibody is labeled with a probe.

Indirect detection is more commonly used. This is where the primary antibody is unlabeled and a labeled secondary antibody binds to the primary antibody. Because of the broad specificity of the secondary antibody, one secondary antibody can be used to detect a wide range of primary antibodies, making this method highly cost-effective.

Difference between Indirect vs. direct detection in Western blotting
Figure 1.3. Indirect vs. direct detection

5. Antibody detection

Depending on the label bound to the antibody, antibody binding can be visualized using colorimetric, radioactive, chemiluminescent, or fluorescent detection methods.

Chemiluminescent Western blotting is a popular indirect detection method that relies on an enzyme-substrate reaction that emits light (Figure 1.4). Horseradish peroxidase (HRP) and alkaline phosphatase (AP) are two enzymes commonly used to label antibodies. The sensitivity of chemiluminescent detection depends on the choice of substrate—commercially available substrates for HRP can detect proteins in the femtogram range.

Depiction of chemiluminescent Western blot signal
Figure 1.4 Chemiluminescent Western blotting- one signal, one protein

Fluorescent Western blotting uses antibodies directly conjugated to fluorescent dyes. Unlike chemiluminescent Westerns, which are limited by the variable kinetics of the enzyme-substrate reaction, the amount of light emitted from fluorophores is highly consistent and directly proportional to the amount of protein on the membrane. This consistency means that fluorescent detection can provide a truly quantitative analysis of protein amount.

Fluorescent detection allows for multiplexing, in which multiple proteins can be detected simultaneously on the same blot (Figure 1.5).

Multiplex fluorescent Western blot
Figure 1.5. Multiplex fluorescent Western

Advantages of Western blotting

Western blotting has several advantages as an immunodetection technique. It is compatible with a wide variety of protein types and detection methodologies. Here are three advantages of Western blotting that you should be aware of:

If you want to start Western blotting but aren’t sure which imager to use, this article outlines the top five features you should consider when shopping for a new gel doc.

Questions Western blotting can answer


Western blotting is a powerful analytic technique, but no technique is perfect for every situation. The Western blot procedure is time consuming and getting started can require optimization.

There are some disadvantages you should be aware of when embarking on a new experiment:

Western blot imaging systems

Today, Western blot detection is no longer limited to the capabilities of a x-ray film in a darkroom. There are many digital Western blot imaging systems available on the market that allow for accurate quantitation of a range of signal intensities in Western blotting.

Both chemiluminescent and fluorescent detection can be documented with a CCD-camera-based Western blotting system, like the Azure Imaging Systems (the only imager on the market to use lasers for higher signal), or through photodiode or APD+PMT detection using the Sapphire FL Biomolecular Imager.

The Azure 600 (Figure 1.5) is the highest system offered in the Azure Imaging Systems product line. It offers laser technology with two IR detection channels, enabling you the ability to multiplex (image more than one protein in an assay).

Two scientists looking at multiplex fluorescent Western blot on Azure 600 Western blot imager
Figure 1.5. The Azure 600 provides accurate and fast chemiluminescent detection, as well as the sensitivity, dynamic range, and linearity needed for quantitative blot analysis.

If your studies require a wide arrangement of application needs, from Western blotting and live animal imaging, to phosphor imaging, the Sapphire FL (Figure 1.6) is your best bet. It offers customizable and user-changeable optical modules, 5–1000µm resolution scans, a Z-plane range from -1.0 to +6mm, five anesthesia output ports for imaging living animals, chemiluminescence detection through the Chemiluminescence Module, and much more.

Changing out modules in Sapphire FL
Figure 1.6. The Sapphire FL features a unique, patent-pending design of interchangeable and customizable laser and filter modules, enabling a virtually infinite number of spectral combinations. A broad range of excitation and emission wavelengths, as well as phosphor imaging, are supported.

If your application needs stop at chemiluminescence, chemiSOLO (Figure 1.7) is also available in the United States and worldwide.

chemiSOLO can easily and quickly image chemiluminescent Western blots by using your phone, tablet, or laptop- without the need for additional software downloads. It’s the first personal Western blot imager of its kind on the market.

Azure chemisolo next to a hand using a mobile device to connect
Figure 1.7. A unique web browser interface allows the chemiSOLO to be controlled by phone, tablet, or PC, without the need to install any additional software.

LEARN MORE: If you want to learn more about the advantages of digital imaging of chemiluminescent Westerns read Why You Should Leave the Darkroom.

Whatever your Western blot needs are, we can help! Learn more about our list of imaging systems hereIf you’re still unsure of which system you need, an Azure Biosystems expert is available to help you choose the right option. Fill out the form on this page and let us know what you’re looking for in a system. That’s it. We’ll match you up with the best fit.

Frequently Asked Questions ABOUT WESTERN BLOTTING

Western blotting involves supplies such as Western blotting paper, membranes, gels, power supplies, gel transfer tanks, and more. View full list >>

Western blotting’s namesake was coined as a nod to its cousin Southern blotting. Its creator Neal Burnette’s lab was located on the west coast. Check out more history behind Western blotting

Western blotting is used to identify one or more specific proteins in a complex mixture of proteins. It is a powerful tool that provides information about the presence, size, and under the right conditions, even the amount of a protein. It is commonly used and often routine in many labs. Read more about Western blotting here

Various equipment is used in Western blotting such as electrophoresis equipment such as the buffer tank, transfer systems, membranes, reagents, and imaging systems. View a complete list of everything you need for Western blotting

SDS-PAGE is the first critical first step in the Western blotting process to successfully detecting proteins. It stands for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and separates proteins by their molecular weight. How SDS-PAGE separates proteins

Most labs use PVDF or nitrocellulose membranes, but other options are available, including nylon or cellulose membranes. Nitrocellulose membranes are ready to use, easily hydrated, and readily bind with proteins out of the box. PVDF membranes are used for Western blotting because they are most robust and sensitive. Which membrane is right for your experiment? 


Nonfat dry milk is a popular blocking agent because it is inexpensive and easy to find. However, milk contains phosphoproteins that can interfere with blots using anti-phosphoprotein antibodies. Milk can interfere with biotin-streptavidin based detection systems. In these situations, a blocking buffer containing BSA may be preferred. Which blocking buffer to use for Western blotting


New to Western blotting? Need to troubleshoot your Western blot?​ Want to brush up on Western blotting best practices? Claim your free Western Blotting eBook!
Instruments for Western Blotting

Laser and CCD-based imaging system for Western blots, gels, tissues, and more

Adjustable 5-1000 µm resolution perfectly captures Western blots, gels, microscope slides, live animals, and more

Portable, personal chemiluminescent Western blot imager controlled by mobile device

Western Blotting Reagents
Featured Publication

This publication uses the Azure c300 to expose chemiluminescent ECL Western blots on a PVDF membrane.

Additional Western Blotting Resources

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