Western blotting is a widely used analytical laboratory technique that is used in molecular biology to detect proteins within a mixture.
During a western blot experiment, samples containing a mixture of proteins are separated based on their size using gel electrophoresis. Although commonly used as a qualitative method to detect and identify proteins, western blotting can also be used semi-quantitatively.
Western Blotting Resources
Sample preparation is the first stage of protein separation using the western blot method.
The number and complexity of the steps involved will vary based on the sample type and the complexity of the protein mixture.
However, there are two main steps involved with separating any mixture of proteins within a sample.
Methods required to extract proteins from samples will range in complexity. Common methods used to extract proteins from a sample include both physical and chemical processes, including sonication, homogenization or denaturation.
Measuring the total protein concentration of samples is also important, as it will impact the quality of results from electrophoresis and detection stages.
Separation of proteins is performed by loading prepared samples to the gel and applying an electric field.
The protein mixture is then separated, based on chemical and physical properties of the molecule. This separation forms distinct bands on the gel for further analysis.