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Western blotting is a widely used analytical technique that can identify one or more specific proteins in a complex mixture of proteins.
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.
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:
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.
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.
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).
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.
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.
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.
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.
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).
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:
This minimizes protein loss that can occur during sample prep.
Samples can be fractionated to determine where a protein of interest is localized, but this is not necessary
This is valid as long as the user has a primary antibody specific for their protein of interest.
A wide selection of secondary antibodies are commercially available that make detection of most primary antibodies possible, like goat-rabbit, etc.
Western blotting is versatile in that it is compatible with many detection methodologies.
Western blots can be detected using:
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.
Proteins often times bind to DNA in the major groove, but there are certainly exceptions. Western blotting can be used to analyze the eluate from an affinity column to see if a specific protein binds to the column.
With multicolor fluorescent detection, Western blotting can be used to assess two or more proteins in a single sample, including comparison to an internal standard.
Western blotting is an excellent way to demonstrate that a protein of interest is present in a sample.
A band detected on a Western blot provides an easy-to-interpret, visible indicator that the antigen targeted by the primary antibody is present. With correct positive and negative controls, a Western blot will answer definitively whether the protein of interest is in the sample at a level above the limit of detection.
Western blotting can provide quantitative data under appropriate conditions, allowing you compare the amount of the protein of interest present across multiple samples on the same blot.
Both chemiluminescent and fluorescent detection can be used for quantitative Western blots.
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:
We get it- carrying out a Western blot requires several steps and can take from a few hours to overnight.
The good news is there are alternative approaches to various steps in the Western blot protocol (gel running, transfer, antibody incubations, detection) that an speed the process.
Western blotting involves a multi-step protocol and each step may need to be optimized for a successful experiment. Here are some things to consider:
Once these conditions are determined, you will find that Western blotting is a robust and reproducible technique.
In most Western experiments, only one protein can be assessed on the blot. With multicolor fluorescent detection, two proteins (or potentially three or four, depending on the imaging system) can be detected in one experiment.
Read this to learn why you should multiplex your Western blots.
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).
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.
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.
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 here. If 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.
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
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
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This publication uses the Azure c300 to expose chemiluminescent ECL Western blots on a PVDF membrane.
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