It’s always a great day when the long-awaited results of my western blot come back clear and easy to interpret. My project this summer involves many, many western blots. Western blot is an important technique used to detect proteins in a sample. To start my project, my mentor and I dissected samples of different regions of mice brains at different time points in development from both our mice model of dystonia and its littermate controls. I am now using western blot analysis on these samples to determine if there is a dysregulation in the levels of phosphorylated eIF2alpha (our protein of interest) when comparing the mice with dystonia to the normal mice.
Though the process of western blot remains the same, my day to day in lab changes based on what step I am at in the process. The whole process of western blot from start to finish typically takes about 3 days. Though it takes a while to get results, it is very rewarding when it works correctly. The process begins by first homogenizing the brain tissue samples and adding buffers to ensure the proteins remain in tact. This step is done in the cold room to ensure the proteins don’t denature. I then do a BCA analysis to determine how strong the protein concentration is in my sample, so I know how much sample to use when I run the gel.
The next step is gel electrophoresis, which helps separate the proteins based on their size using an electric current. After the gel is run, the gel is then blotted onto a solid support membrane to further analyze the proteins. In order to prevent nonspecific binding of the antibodies to the membrane, I add a blocking buffer to the membrane to block out any nonspecific spots on the membrane. To visualize the protein of interest, I then probe the membrane with a primary protein-specific antibody. The primary antibody binds to the protein of interest like a lock and key. I then probe the membrane with a labeled secondary antibody used for detection. I then use imaging to detect the protein-antibody-antibody complex on the membrane. Finally, I analyze the results to ensure the presence of a protein of interest, the amount of protein, and its size.
Now that I am finally starting to understand the process and complete it primarily on my own, I come into lab everyday excited to learn from a previous blot. I love having the ability to implement better technique each time I complete the process. In addition, I am constantly learning new and better ways to complete each step from my lab mentor and other members of my lab. My lab has been incredibly welcoming and always willing to answer my questions. Though the process of western blot can seem a bit tedious at times, I am grateful to have the opportunity to keep learning through the process and be surrounded by such a supportive lab environment.