The Western blot workflow for immuno-based protein detection was introduced over 30 years ago. Since its introduction, Western blotting has become a well-known and widely accepted technique routinely used in many laboratories to detect a specific protein from a complex mixture. Because of the unambiguous results and wide scientific acceptance, Western blotting is often used to confirm results generated from more complex experimental techniques.
Many advancements have improved the convenience and performance of Western blotting. These advancements include the transition from radioactive to chemiluminescent detection, the transition from using homemade polyacrylamide gels to pre-cast gels for electrophoresis, and, most recently, the transition from using bulky and wasteful tank-transfer devices to the more rapid semi-dry transfer devices. Yet, despite these significant advancements, the Western blotting workflow remains a time-consuming and labor-intensive process with many steps that often require optimization.
To perform a typical Western blot, proteins are separated by gel electrophoresis and transferred to either nitrocellulose or PVDF membranes. The nonspecific binding sites on the membrane, where no protein was transferred, are blocked by incubating the membrane in proteinaceous blocking buffer. Because of its low cost, 5% milk is a popular blocking buffer. Protein detection on the membrane begins with primary antibody incubation followed by wash steps to remove nonspecifically bound antibody. Unfortunately, not all primary antibodies have equivalent performance.
Some primary antibodies generate nonspecific bands (i.e., bands other than the protein of interest), and some require an overnight incubation to obtain adequate detection sensitivity. Empirical testing to determine the optimal concentration is often required. Using an insufficient amount of antibody results in poor signal detection and too much antibody can cause high background or a specific signal that is too intense.
After the wash steps are complete, the membrane is incubated with an enzyme-conjugated species-specific secondary antibody, which is followed by more washes and signal detection. A variety of chemiluminescent detection reagents are available for different sensitivity levels.
The concentration of secondary antibody is a critical step in the process and will vary depending on the substrate of choice. A frequent error is the use of the secondary antibody at a concentration that is too high, which can result in detection of nonspecific proteins, high background, or in some cases, inadequate signal from rapid depletion of the chemiluminescent substrate. Extensive washing is required to remove the nonspecifically bound antibodies and can be essential to reduce background and improve the signal-to-noise.