Scotty McKay: The Life of a Protein

I worked in Dr. Marian’s lab for three semesters. During this time, I learned to culture cells, determine the protein concentration of a solution, perform immunoprecipitations, perform immunofluorescent microscopy, SDS PAGE, as well as western blotting. I was also able to present my work at three different conferences and wrote my honor’s thesis titled “Cadherin 18 Localization and Interacting Partners” over this project. I got into this research because I wanted to explore whether research was something that I would want to pursue after graduation, but also because I saw potential for this work to have a real, positive impact on people’s lives.

The goal of this project was to learn more about a protein that is found naturally in human cells called cadherin 18. This is a type 2 cadherin that very little is known about, which to me is one of the most fascinating things about this project. Knowing throughout your research that something you notice could be the first time that anyone has ever made that observation is an awesome feeling. For this project, we had two main goals. These goals were to find out where cadherin 18 is localized within cells as well as determine what proteins cadherin 18 interacts with. By knowing where it is located and what proteins it interacts with, we will have a much better idea of what it does in the cell. Gaining a greater understanding of cadherin 18 is important because by learning more about cadherin 18, we will have a better understanding of any bodily process or disease that cadherin 18 is found to be involved in.

In order to see where the cadherin 18 is located in cells, we used immunofluorescence. Immunofluorescence makes use of antibodies as well as fluorophores (light emitting organic molecule) to locate a particular antigen. In our case, we bound a primary antibody that specifically binds cadherin 18 to cadherin 18. We then bound a secondary antibody to the primary antibody. This secondary antibody had an attached fluorophore that emitted light when it was exposed to a certain wavelength of light. Using this method, we were able to take pictures of our cells that highlighted where cadherin 18 is located.

We then used immunoprecipitation to determine what proteins cadherin 18 interacts with. Immunoprecipitation uses the same antibodies as immunofluorescence that are specified to cadherin 18. In this case though, the antibodies are bound to agarose beads. After the cell’s proteins are extracted, all the cellular proteins are allowed to interact with the antibodies. Only the cadherin 18 should bind to the antibody, and any protein that interacts with cadherin 18 should bind to cadherin 18. After performing washes and centrifugations, we should be left just with the beads, antibodies, cadherin 18, and possible interacting partners of cadherin 18. An elution buffer was then used to remove the proteins from the antibodies, and the proteins were subjected to an SDS PAGE to help determine what the interacting proteins are. During SDS PAGE, the eluted proteins are mixed with a dye and then pipetted into the wells of a precast gel. This gel is then put into a tank of SDS buffer which denatures the proteins and gives them a negative charge. When this gel is subjected to electrophoresis, the proteins are pulled toward the positive electrode. The gel that the proteins move through is very porous which allows small proteins to move more quickly through it. The end result is a gel with multiple bands of proteins separated based off molecular weight.

The end result of my work on this project is that we believe, based on our fluorescent images, that cadherin 18 is located throughout the cytoplasm of cells. We also were able to see multiple distinguishable bands on our SDS PAGE which we believe are possible interacting partners of cadherin 18. This project will be taken over by another student next semester who will help verify and expand upon these results. I am very happy that I was given the opportunity to assist with this project and am extremely pleased with the results we were able to get in such a short amount of time. I highly recommend that other students get involved in some research project of their own. This was an experience that not only taught me useful skills, but also an experience that greatly developed my critical thinking, problem solving, and presentation skills which will be invaluable in the future.

 

Speak Your Mind

*