Western blotting is a widely used analytical technique that can 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. Though commonly used and often routine in many labs, Western blotting can be source of frustration when it doesn’t work. It involves several steps, each of which needs to be optimized to achieve the best results. The key to the best Westerns is understanding the process. Below is a brief overview of each step. You will find more detail as well as optimization tips in our free Western Blotting eBook, How-to Guides, and Application Notes.
In the first step of a Western blot, 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, which are negatively charged under the experimental conditions, 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 blots include nitrocellulose 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 Western blot data, especially when quantitative information is desired. 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 ready to be probed with antibody and unbound antibody washed away. The factors that influence probing and washing include whether a direct or an indirect detection method will be used (Figure 1.3), the quality and type of antibodies available, the number of antigens to be detected, the type of enzyme or tag that will be used for detection, and the incubation and wash conditions. The indirect detection method is more popular and often more sensitive than direct direction. With indirect detection, the antibody that recognizes the antigen of interest, called the primary antibody, is unlabeled while a secondary antibody that binds to the primary in a second incubation step is labeled with an enzyme or fluorescent probe. 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.
Antibody binding can be visualized using colorimetric, radioactive, chemiluminescent, or fluorescent detection methods. This guide focuses on chemiluminescent and fluorescent detection. The choice of detection method should be made based on multiple factors including the desired sensitivity.
Chemiluminescence is a popular indirect detection method for Western blotting that relies on an enzyme-substrate reaction that emits light (Figure 1.6). Horseradish peroxidase (HRP) and alkaline phosphatase (AP) are two commonly used chemiluminescent enzymes, with the sensitivity of detection dependent on the choice of substrate—commercially available substrates for HRP can detect proteins in the femtogram range. Imaging of a chemiluminescent Western blot is historically done via exposure of the blot to x-ray film or using a CCD-based imaging system.
Fluorescent Western blotting uses secondary 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 proteins amount. Fluorescent detection also allows multiplexing, in which multiple proteins can be detected simultaneously on the same blot (Figure 1.9).
Looking for a full list of applications?
Related blog posts…
Validating your antibodies, i.e. confirming that an antibody recognizes your protein of interest with low cross-reactivity to other targets, is critical for ensuring consistent, reproducible