Western Blotting成功宝典

Introduction

Western blotting, also called immunoblotting, is a widely used and accepted technique to detect levels of protein expression in a cell or tissue extract. This technique measures protein levels in a biological sample through antibody binding to a specific protein of interest. The precise binding that occurs between an antibody and its target protein epitope allows detection of highly specific amino acid sequences within a protein. Antibodies can also detect specific post-translational modifications (PTM) of a protein. Phospho-specific antibodies have been used to identify components of specific signaling pathways and to study changes in phosphorylation events in various biological contexts. Antibodies specific to other PTMs have been developed recently, allowing researchers to monitor changes in acetylation, methylation, and ubiquitination status of a protein.

At Cell Signaling Technology (CST), scientists perform more than one thousand western blots daily to validate our existing and new antibodies. The western blot protocol we have been optimizing for over a decade can be seen on page 13 and is also available online (see link below) so you can replicate the procedure and get reproducible and reliable results.

Here, we will highlight the critical steps in the western blot protocol and demonstrate how protocol changes can affect the final outcome of your blot. We will also discuss the importance of using a well-validated antibody in your western blot experiments. Finally, we will provide a list of commonly used reagents that are utilized by CST scientists and work optimally with our antibodies.