This week started out great, with the Spurs winning on Sunday beating the Heat. Just when I thought my coworkers couldn’t be any happier or nicer, I am pleasantly surprised. The lab looks cleaner, the glassware is shining, and the hallways smell like victory in place of the familiar urine, iron, and formaldehyde-blended scents. This week I’ve been back and forth between two lab benches on opposite sides of the building, working on the preliminary data that measures the activity of cannabinoid receptors, specifically CB1 and CB2, in the collected lymphocytes.
Working in vitro I have had to supply the lymphocyte samples (they look like miso soup before sonficiation, see Figure 1.) with ATP and a compound called Forskolin that stimulates the Adenylyl Cycalse partnered to the G-protein. This lets me know that everything is in working order in the cells and confirms their ability to produce cyclic AMP (cAMP). The next step will be to use a full agonist (whose name I will withhold, sorry!) to target cannabinoid receptors.
Keeping up with two separate lab benches is tough work. I basically depend on sticky notes that I attach to my lab notebook and workspace to remind me of everything I need to take to and from one bench to the other, as well as any specific instructions. It feels a lot like Memento if you substitute the action scenes for pipetting/decanting/ etc. and the polaroid photographs with updates from my mother and father about my cat – and how their vacations in Hawaii and Vermont are panning out. As a refresher, the ELISA assay kit I am using is measuring the cAMP concentrations produced by the CB1 and CB2 receptors, this way we’ll get a feel for the activity of the receptors in blood. This week I am stimulating the G-coupled protein receptor component of these CB-R’s using Forskolin, but this process bypasses the CB1 and CB2 receptors. I’ll be working on binding to the actual receptors of interest using a synthetic analgesic agonist next week.
The first experimental run came back totally flatlined. Still a bummer, even after everyone told me to expect it for the first five trials. No amount of cAMP was detected by the machine. This could have been for many reasons, so I had to go back to the drawing board with my bosses. Culling through all the literature related to Forskolin, cAMP, and Adenylate Cyclase we finally discovered one of the many probable reasons for lack of cAMP. The incubation buffer used was sterile platelet buffer, having no Mg2+ or Ca2+ ions. Ions are needed to facilitate certain substrate bindings since they play a large part in generating the affinities between an enzyme and its associated substrate(s). I’m currently running the same experiment again, waiting for different, hopefully better, results.
Figure 1. Sonicator - cell disrupter. I could have shown you the miso soup lymphocyte samples, but honestly it would just make you hungry. Instead I present the sonicator that I have been using this past week to homogenize samples. I am terrified of this machine. It applies sound energy to break apart and disrupt cell membranes, also homogenizing them. If you were to hold your finger against the tip and just ever so slightly press up, the ultra sonic frequencies would drill through your finger. It also makes some seriously annoying sounds.
Moving into a more translational side of the study, the PI’s overseeing this project have decided to use mice to look at the amount of CB1 and CB2 receptors in regions of the brain. I’m studying brain atlases and tracking down all the tools I will need for the extractions in preparation for the mice arriving next week. I wasn’t expecting the opportunity to get my hands dirty like this again until next Fall semester. I am stoked.
Figure 2. HPLC results measuring alcohol in blood samples taken from coworkers. We’re all friends here.
Ocha & Wagashi
What I have learned or been reminded of so far:
1. I actually love anything to do with brains, and can’t wait to start working with mouse brain tissue.
2. I don’t enjoy the thought of having to putting down animals.
3. The olfactory bulb takes up a rather large amount of space at the front of a mouse’s brain, go look at it: http://www.hms.harvard.edu/research/brain/atlas.html