Western blot analyses are run to separate and identify proteins. First, the proteins are separated through gel electrophoresis. The proteins are transferred to a membrane where they are marked with antibody, and a film is developed for visualization (‘Western Blot: Technique’).
Waiting for the gel to solidify:
Adding the samples of Rat liver homogenate into the gel:
Waiting for the gel to run:
At the beginning:
Here, the protein is being pushed through the gel by an electric field. The purple dye is used to identify the edges.
The next step is blotting, where the proteins on the gel are transferred to membrane paper using a semi-dry cell.
After waiting for an hour and a half, the membrane is stored in the cold room overnight at 4 degrees Celsius. The membrane can also be stored at room temperature for an hour.
In the morning, I cut the membrane in half and pour two different antibodies onto each.
These are antibodies oatp1 and ae745.
I placed them on the orbital shaker for two hours.
I proceeded by washing away non-specific proteins!
I run antibody 2 with Rabbit HRP and performed a second round of washing.
Lastly, I develope films of the western blot analysis in the film room.
Here is the final result!
The black lines are where the antibody bound to the protein. Since they are clearly visible, this western blot analysis was a success!
I would like to give a special thanks to Peggy Wang for mentoring me throughout this process, and to Dr. Allan Wolkoff for providing the opportunity to work in his lab!
Mahmood, T., & Yang, P. (2012, September). Western Blot: Technique, Theory, and Trouble Shooting. Retrieved July 21, 2017, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3456489/