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.
The first challenge was finding the protocol for protein extraction. In order to study the cytochromes of the rat liver, I will need to grind up the tissue and extract the protein from there. Finding actual protocols for how to do the kind of protein extraction I want was difficult as there are many factors about the tissue and what supplies I have available that go into the extraction. Even once I found a protocol here, the process is by no means simple, and requires several steps detailed below.
The next challenge in terms of separating the proteins of interest is in electrophoresis. This is a process that uses an electric current to separate the proteins based on their polarity. Finding a protocol was not entirely difficult, as it is a fairly common procedure, but finding one specifically for the cytochrome p450 group is proving to be a challenge, so we are simply trying to find sources with enough material to help us further our research, such as one here.
The basic process of electrophoresis is detailed below:
After the electrophoresis separates the proteins, the next step is analyzing the quantities of the various proteins extracted. This is very important to our specific experiments and is done in a process called western blotting. However, this is a complicated enough process to merit its own post.
So, until next week, that’s all folks!