The western blot (WB) western blot service is a common method for analyzing protein samples. The macromolecules in a sample are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis according to their molecular weight and then transferred to a nitrocellulose or polyvinylidene difluoride membrane, where the target protein is detected by antibodies that recognize specific epitopes on the protein. In most cases, the target antibody is tagged with a molecule that produces a detectable signal when coupled to a labeling enzyme, such as horseradish peroxidase or alkaline phosphatase. The labeled antibody is then bound to a chromogenic or fluorescent detection system that allows the signal to be visualized.
A key factor in interpreting a WB is the linear and proportional relationship between the amount of protein extract loaded on the gel and the signal intensity observed on the membrane. Choosing an internal loading control that is present at a relatively constant level across all experimental conditions is essential for this relationship to hold. Loading controls can be housekeeping proteins, single tag labeled antibodies, or total cellular protein. Serial dilutions of the experimental sample can help to identify an appropriate combined linear range of detection for both the target protein and the internal loading control.
Monoclonal Antibody Service: Streamlined Development and Validation
The sensitivity of a Western blot depends on how much protein is loaded on the gel and the detection system used to visualize the protein bands. Whether the Western blot is to be visualized by chemiluminescence or fluorescence, the exposure time required can limit the signal-to-noise ratio of a band. To avoid the need for a dark room, a film developer or a fluorescent reader, the most popular approach to protein visualization is with a WESTERNVIEW® Detection Kit that includes both the secondary antibody and all the reagents needed to generate a visual readout within minutes without the use of specialized equipment.