Western Blotting

This past week I have been working with bacteria, to ultimately produce a western blot, which detects specific proteins in a sample of many different bacterial proteins, and provides a comparative quantity of protein between the different strains of bacteria. In the western blot we want to detect the LasR protein, which is a receptor in quorum sensing in the bacteria.

There are many steps involved in a western blot that ultimately allow you to be able to see your protein. After we grew our bacteria and isolated the proteins, we could begin the first major step in creating a western blot- Polyacrylamide Gel Electrophoresis. We made a gel containing wells where we would then insert our samples. An electric current is then run through the sample, causing the proteins to separate based on size. Larger proteins move slower in the gel, and therefore they will be closer to the starting point than smaller proteins when the electric field is shut off. From the gel, we then transferred the proteins onto a membrane that was soaked in methanol, which promoted interaction with the negative charged proteins.

After successfully moving our protein to the membrane, we added milk to prevent nonspecific binding of proteins. We could then add the primary antibody, which specifically targets the Las R protein, however it is possible that this antibody could bind to other proteins as well. In order to be able to see the LasR protein we added a secondary antibody, which binds to the primary antibody and contains an enzyme that illuminates when exposed to a specific substrate.

Finally at the end of the week, we worked to develop the blot so we could visibly see our protein. In a dark room we added substrate and transferred our proteins onto a light sensitive film, which was then submerged in developer, water, and then fixer. However at the end of this process there were no bands clearly visible. After talking with my mentor we came up with several possible reasons why our film may not have developed properly, however with so many steps it can be difficult to identify the issue. Next week we will retrace our steps to hopefully identify the problem, and develop another western blot.

Citations

Western Blotting Fundamental. https://www.bosterbio.com/protocol-and-troubleshooting/western-blot-principle (accessed July 21, 2017).

Table of Contents https://starcellbio.mit.edu/static/ref_lib/full_library.html (accessed July 22, 2017).