For my on-going project, as discussed in my previous blog entry, I use the BRET assay to measure protein-protein intracellular interactions specifically by investigating the role of arrestin plays in internalization and signaling of the D2 dopamine receptor depending on the location of the receptor. Now, this assay can be modified in several manners, for example, to manipulate concentration of certain g-protein coupled receptor kinases (GRKs) or to observe the effects of different ligands or drugs in activating the receptor. Essentially, this assay can be and will be modified in a million and one ways using a million and one differing conditions, excuse my hyperbolic tone, to further uncover the mechanisms of dopamine receptor signaling and trafficking. With that being said, you best believe I have a million and one different BRET experiments that I have to conduct. So here is how the deal basically goes:
- Monday means transfections. My mentor graciously comes on the weekend to have cells already split and ready for me to transfect on Monday with the certain receptor and protein tags. Thanks Tom, you rock for that.
- Tuesday means splitting. After I have transfected the cells, they must be split onto a special 96-well plate in specific media. Multiple transfection plates usually means I have a long afternoon in the cell culture room.
- Wednesday means treatment. I treat the cells on the 96-well plate with different drugs for an hour and then I can read the plate using the lovely BRET machine.
- Thursday means repeat. Usually, I have several BRET assays staggered so I will transfect, split, and read all through the week.
- Friday means analysis. This is the day I go through all the data I have collect and begin to interpret the results. After, I can plan out where I will go from there and what conditions I will use for the next BRET assay.
In addition to my BRET project, I am usually kept busy helping my mentor with his own experiments. Cloning, cloning, and more cloning. But I am not complaining. I love working in the Caron lab. I love that I always have something to do and I am never bored. I love the repetition as well as the novelty. I love the science behind everything and the fact that I am learning more and more everyday. Most of all…I love my lovely new friend named BRET.