One aspect of my internship this summer is a project that I will present to the lab staff in August. I have been informed that the only requirement of this project/presentation is relevancy to a current or prior research conducted by the HPL (Human Performance Lab). Because both heat related studies are ongoing, I have decided that, while I continue to help out with them, I will start focusing my attention on a recently concluded HPL study looking at the relationship between cortisol and obesity and start developing a presentation based on this research. Since this study has already been concluded and all samples have been collected, it presents the perfect opportunity to do a little original research (under Joel’s guidance). I expect more specifics next week but until then, I can tell you that we are going to be looking at GR (glucocorticoid recceptors) in plasma samples from the cortisol/obesity study. GR was proposed as a logical candidate for follow up research because it is an important cortisol receptor. Right now, our main objective is to isolate and characterize our GR receptors. The appropriate ELISA kits have been ordered and we started our first receptor isolation dry run today using a western blot to isolate and quantify nuclear ER (estrogen receptors) from blood I was able to get from a team member. (People are remarkably generous with their blood here). Hopefully I will be able to get a couple of western blot dry runs in before the actual kits arrive so I will be proficient enough to do them without Joel’s supervision. I said we started the western blot today because Joel told me that one of the best ways to reduce background is to incubate membrane and blocker (5% milk in this case) for 24hrs, which means we will be able to develop the membrane on Monday or Tuesday. The gel itself ran beautifully! Since we have been given the freedom to fine tune the protocol, we decided to try two methods of cell lysis: freeze thaw (3x) and sonication in order to break the ER free from the nuclear membrane. I must say the freeze thaws were particularly fun because we used dry ice to “snap” freeze the the samples after each thaw. And what a difference! the sonication lanes were almost clear while the freeze thaw lanes were muddles with cell debris! The transfer to the nitrocellulose membrane very quickly (7 minutes) using a new transfer machine although the gel marker lanes still has some visible bands, which might have indicated that not all the proteins were transferred to the nitrocellulose. We then submerged the membrane in milk for blocking in order to prevent non-specific binding of our antibodies and prepared the primary and secondary antibodies for development after the weekend. We are hoping that if we get clean results, we can use a similar protocol in the coming weeks with the new kits to isolate and quantify GR.
Other than working on the western blot, this weeks highlights included two presentations on the the effects of preventing the dimerization of GR in mice and my first real lab meeting, where everyone shared progress reports on whatever they had been individually working on this week and we discussed a protocol issue with the heat tolerance study. Apparently the ASCM guidelines meant to screen for diabetes mandate no food intake for 8hrs prior to the measuring of fasting glucose levels, a key biomarker collected prior to the VO2 max test. The issue, as I understood it, was that subjects’ max scores might be significantly influenced by this rule. In the end it was decided with the input of one of the lab physicians that fasting glucose levels could be accurately measured after only 4 hrs of fasting and that stressing our subjects to the fainting point was not necessary.