The incubation step in western blotting

 Western blotting is one of the most reliable procedures for identifying a protein(s) in a mixture of other proteins. It involves eight steps to separate and visualize the protein of interest, they include;

• Sample preparation

• Gel electrophoresis.

• Protein/membrane transfer.

• Blocking

• Incubation with primary antibody.

• Secondary incubation

• Detection.

• Analysis.

The incubation step is one of the most crucial procedures in western blotting. It occurs after the blocking stage and before final detection and visualization. Ensuring thoroughness during this step is vital to the success of the procedure. In this blog, you will learn about the incubation phase and the required criteria for successful immunoblotting.

Incubation is a two-part process that involves the use of primary and secondary antibodies. In simple terms, the primary antibodies bind to the protein of interest. To detect the proteins, the secondary antibodies need to bind to the primary ones. Some factors that affect the quality or sensitivity of the final results include the incubation time, operator handling, reagents and materials used, and the primary antibody concentration for the western blot.

Choice of antibodies.

Scientists use polyclonal, monoclonal, and recombinant antibodies for western blotting. Antibodies are created using the natural immune response of a host species. For example, when a foreign entity like bacteria or virus enters your body or any other organism, immune cells create specific proteins that bind and destroy the alien or attract a macrophage to digest it.

Western blotting applies this same principle to create highly-specific antibodies.

The secondary antibodies, on the other hand, attach to the primary antibodies. They are conjugated with an enzyme such as Horseradish peroxidase, a radioactive label, or a fluorescent antibody.

Blocking prevents the antibodies from binding with the membrane, leaving them with no option but to bind with the appropriate antigen. Washing is crucial before and after every step of the incubation process to remove the unbound proteins, antibodies, and antigens to minimize artifacts or noise in the final results.

Incubation time for western blotting.

The Primary antibody incubation time largely depends on the binding affinity to the target protein. Common protocols for incubating with the primary antibody placing the membrane in the antibody-buffer solution for 1-2 hours at room temperature or overnight at 4 C- The same rule applies to secondary antibody incubation.

Conclusion.

The incubation step in immunoblotting is a vital part of the process. However, many factors can contribute to inaccuracy or challenges when analyzing the results. These errors could occur during the incubation and pre/post incubation stages.

Advancements in biotechnology have provided innovative solutions to some challenges faced in the western blotting process. Take for example, automation and one-step probing. Automation minimizes errors by eliminating human errors during handling, and time, while improving reliability and reproducibility.