Western Blotting

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. The key to the best Westerns is understanding the process. Below is a brief overview of each step.

Gel electrophoresis for Western blotting

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

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Quick guides to Western blotting

Figure 1.1. Polyacrylamide gel electrophoresis

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 setup
Figure 1.2. A typical semi-dry transfer setup

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.

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 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.

Membrane incubation with antibody

After blocking, the membrane is probed with antibody and then unbound antibody washed away. For direct detection, the primary antibody is labeled with a probe (Figure 1.3). More commonly, indirect detection is used in which the primary antibody is unlabeled and a labeled secondary antibody binds to the primary antibody (Figure 1.3). 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.

Figure 1.3. Indirect vs. direct detection

Antibody detection

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

Chemiluminescence is a popular indirect detection method for Western blotting 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.

Figure 1.4. Chemiluminescent Western blotting- one signal, one protein. In chemiluminescent detection, the antigen-primary antibody complex is bound by a secondary antibody conjugated to an enzyme, such as horseradish peroxidase (HRP). The enzyme catalyzes a reaction that generates light in the presence of a luminescent substrate, and the light can be detected either by exposure to x-ray film or by a CCD-based imaging system.

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 can be documented with a CCD-based system such as the Azure Imaging Systems or photodiode or PMT detection such as the Azure Sapphire. Fluorescent detection allows multiplexing, in which multiple proteins can be detected simultaneously on the same blot (Figure 1.9).

Figure 1.9 Multiplex fluorescent Western


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