Western blot
Introduction
Western blotting, also known as protein immunoblotting, is a widely used analytical technique in molecular biology, biochemistry, and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract. This method uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide. The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are stained with antibodies specific to the target protein.
Procedure
The process begins with the preparation of a protein sample, which is then loaded into a gel for separation. The gel is subjected to an electric current, causing the proteins to migrate through the gel. The proteins are then transferred onto a membrane, which is then incubated with a primary antibody specific to the protein of interest. The membrane is washed to remove excess antibody, then incubated with a secondary antibody that is linked to an enzyme. The enzyme catalyzes a reaction that produces a visible signal, indicating the presence and location of the protein of interest.
Sample Preparation
The first step in a Western blotting procedure is to prepare the protein sample to be analyzed. This typically involves lysing cells or tissues to release the proteins, and then denaturing the proteins by boiling them in a detergent that breaks the proteins' tertiary structure. The protein concentration is then determined, usually by a Bradford assay, and the sample is mixed with a loading buffer that contains a tracking dye to monitor the progress of the electrophoresis.
Gel Electrophoresis
The protein samples are loaded into wells in a gel, typically a polyacrylamide gel. An electric current is applied across the gel, causing the proteins to move through the gel matrix. Smaller proteins move faster through the gel than larger proteins. The result is a gel with proteins separated by size, with the smallest proteins at the far end of the gel and the largest proteins near the wells where the samples were loaded.
Transfer
The separated proteins are then transferred from the gel to a membrane, typically made of nitrocellulose or polyvinylidene difluoride (PVDF). The transfer is accomplished by applying an electric current across the gel and membrane, which drives the proteins out of the gel and onto the membrane. The proteins bind to the membrane, where they can be probed with antibodies.
Probing
The membrane is first blocked with a protein or nonionic detergent to prevent nonspecific binding of the antibodies. The membrane is then incubated with a primary antibody that is specific to the protein of interest. The primary antibody binds to the protein, and the membrane is washed to remove any unbound antibody. The membrane is then incubated with a secondary antibody, which is linked to an enzyme. The secondary antibody binds to the primary antibody, and the membrane is washed again to remove any unbound secondary antibody.
Detection
The enzyme linked to the secondary antibody catalyzes a reaction that produces a visible signal. The most common enzymes used are horseradish peroxidase (HRP) and alkaline phosphatase (AP), which catalyze reactions that produce light or precipitate a colored product. The signal can be detected and quantified using a variety of methods, including chemiluminescence, colorimetric detection, and fluorescence detection.
Applications
Western blotting is used in a wide range of applications, including the detection and quantification of specific proteins, the determination of protein size, and the analysis of protein expression and post-translational modifications. It is a common technique in research laboratories, and is also used in clinical diagnostics to detect proteins associated with certain diseases, such as HIV infection and Creutzfeldt-Jakob disease.
Limitations
While Western blotting is a powerful technique, it does have some limitations. It requires a relatively large amount of protein, and the procedure is time-consuming and requires careful optimization. In addition, while the use of antibodies provides specificity, it also introduces the possibility of cross-reactivity and nonspecific binding.