After extensive preparation and reading, I am nearly at the point where experimentation will begin. I, like my classmates, am entirely new to the type of lab work going into this course, so even the most essential procedures are new territory for me. In order to help prepare for extensive work of a scope I’ve never before done, I have made my best efforts to be proficient in the material and methods that will be used for the western blotting. Hopefully, this will lead to experimentation soon after thanksgiving break.
As I mentioned in my previous blog post, this week’s post will be dedicated to explaining the process of western blotting.
First, you must understand the reason one would need to know how to do a western blot. The reason is actually quite simple. In any given tissue sample, if the concentrations of various proteins needs to be know, then a western blot is your weapon of choice. Essentially, through a several-step process, a western blot lets you see the quantities of a certain protein in a given substance.
Now that I am beginning to move towards the actual experimentation, it is important for me to develop a plan for what actually has to happen in the lab. The largest part of this is the creation of protocols. Protocols are essentially the recipe for the experiment that is being undertaken. This includes the materials and chemicals needed, as well as the process of the actual experiment. However, finding protocols for the several experiments I selected may turn out to be more difficult than it sounds.
While it is important to understand the fine points of the research I am doing, it is also essential to understand the long-term goals of the research. I met with the head of the mice research this past week at the Coatesville department of Veteran Affairs.
Since the area I am exploring is relatively untouched in the scientific world, it is often difficult to gain a background understanding of the interactions that occur between morphine and the opiate detoxification enzymes. Thankfully, I was able to help gain an understanding of the interactions present in the liver through two papers that analyzed the interactions between the critical cytochrome enzymes and methadone, a synthetic opiate. Though methadone is not identical to morphine, the basics of the interaction are similar enough to get me a little extra important knowledge going into the actual experiments.
The process, in simple terms, begins with the introduction of methadone into bloodstream, which in turn deposits in the liver. The methadone, which attaches to opiate receptors, is then sent to the hepatic portal circulation, where the hepatocytes check the blood for potentially harmful contents. The toxic contents of the liver are then cleaned out and neutralized by enzymes. The cytochromes that deal with methadone specifically are CYP2B6, CYP3A4, and to a lesser degree, CYP2D6.
During this year of independent study, I will be working on studying the effects of habitual morphine use on the livers and kidneys on mice. I will be working with an outside lab to examine the organs of mice that were given steady doses of morphine during their lives. We will specifically be looking at the effects of the morphine on the enzymes known as Cytochrome P450. This is a large family of enzymes with a range of purposes, but CYP3A4 and CYP2B6 are the specific enzymes that deal with the detoxification of the blood in the liver and kidneys. Specifically, these enzymes work to neutralize opiates, both synthetic and otherwise.
The liver, which processes and cleans the blood.
To begin, I researched the background information necessary to understand how the liver processes the blood and toxic substances. I came to an understanding of the various ways in which the liver is damaged by the drug in the long term, which can be found here.