By Dr. Joanne Elliott

Western Blotting has been a common technique in molecular biology laboratories for more than 30 years. This mainstay technique uses gel electrophoresis to separate native or denatured proteins in a tissue homogenate or protein extract. These proteins are then transferred onto a nitrocellulose or PDVF membrane, which can then be stained in a solution containing antibodies directed against the protein of interest. After staining with the primary antibody, the membrane is washed and incubated in a solution containing a secondary antibody linked to a reporter to enable detection. During detection the reporter (such as HRP) can cleave a chemiluminescent agent to produce luminescence. The amount of luminescence produced reflects the abundance of protein when normalized to a protein standard that should remain constant between samples.

The technique can provide researchers with information about a protein’s molecular weight, abundance, as well as expression pattern. Western Blotting can also be used to study post-translational modifications such as phosphorylation or protein-protein interactions. Today, Western Blotting is still the definitive diagnostic test to confirm HIV, BSE (Bovine Spongiform Encephalopathy), as well as some forms of Lyme disease, Hepatitis B and Herpes Simplex virus infection.

Three independent research groups in Seattle, Stanford and Brasil all deserve credit for developing the methods we use today to detect specific proteins using antibodies. However, apart from advances in Western Blotting detection methods and other small tweaks, the common principle of the Western Blot has remained unaltered since publication in 1979. Although Western Blotting is a tried and trusted technique, the method has many disadvantages. The entire procedure is very time consuming and manually cumbersome. Moreover, Western Blotting requires micrograms of protein and the number of samples that can be analyzed is often defined by the size of the gel. In an attempt to address these problems, researchers at UC Berkeley led by Amy Herr, have tried to reinvent the Western Blot. They recently published their work entitled “Single Cell Western Blotting” in Nature Methods in June 2014. The group developed a microscale Western Blot that can be used to analyze approximately 2000 individual cells in a microscope slide format. The technique starts by applying a suspension of single cells onto the polyacrylamide gel containing microwells. Once excess cells are washed away, those that have settled into the wells are rapidly lysed and solubilized within 10 seconds. An electric field is then applied across the slide to drive the proteins through the walls of the microwell. Proteins are separated within 500μm before UV light is used to covalently attach the proteins to the gel. Immunostaining with a primary and secondary antibody can then occur before the protein of interest is analyzed using a standard fluorescent microarray scanner.

This new method certainly has some advantages compared to the traditional Western Blot. Since individual cells can be studied, issues of heterogeneity in protein extracts are eliminated. In addition, the technique requires only 0.01 to 0.5 micrograms of protein which means protein characterization within rare cell types is achievable. The rapid cell lysis and protein separation also eliminates the laborious time consuming steps of a traditional Western Blot. The inventors are currently collaborating with industrial partners to commercialize their single cell Western Blotting technique. However, time will tell if researchers are ready to adopt this new approach to protein analysis.