Preparation for an SDS-PAGE Gel

Last Friday (September 6th), I went over to RPI for the first time during this school year! My primary job for the day was preparing for an SDS-PAGE gel. For this gel, we are working with a specific protein of interest.Ultimately, the gel will analyze the bands of the null solution (which does not contain the protein of interest, but contains other host cell proteins (HCPs)) versus the bands of the solution that contains the protein of interest (in addition to the other HCPs). To do this, we are working with different types of protein solutions. First, there are the null solution and the protein of interest solution which are both prepared at 1X and 10X concentration. The different concentrations will ultimately lead to different width bands. Also, there are 10X desalt solutions for both the null and protein of interest. In the original solution, the buffer contains metal ions that can affect how the bands are displayed in the gel, making them appear blurred. In the desalt solutions, the buffer has been exchanged to PBS to eliminate the metal ions and get cleaner bands. Once the gel is run, we expect to see something like the simplified gel below. We expect to get certain bands from the null solution that represent the bands of the HCPs, and we expect to get those same bands from the protein of interest with the addition of an extra band that represents the protein of interest (represented in red in the image).

Today, to prepare for running this gel, I first made the Tris/Glycine/SDS buffer by combining the stock solution with water, mixing the buffer, then putting it on ice to keep it cold (keeping the buffer cold allows for sharper bands in the gel). Next, I made the blue Laemmli sample buffer by combining the stock solution with β-mercaptoethanol (BME). I then labled the solution tubes with the different solutions and added 20 micro-liters of Laemmli buffer to each sample. After each sample was allocated into its respective tube, I denatured the protein solutions in a heat bath for one hour. To prevent the sample tubes from popping open in the heat bath, I wrapped each sample with parafilm (which ultimately caused them to stick together). After the proteins were denatured, I spun the samples, and set them aside to cool. I then set up the gel equipment.

The gel was then ready for JP to run later in the day! I can't wait to see the results when I return to RPI on Friday!

Back to Senior Year!

Although I did not blog this summer, I continued to work in the lab at RPI! Over the summer, I learned so many skills that I can't wait to apply to my work this school year. After my experience at RPI last year, I could not be more excited to start another year in the same lab. Spending an extended period of time in the lab has allowed me to continue to practice different skills with the machinery and materials in the lab, so I am now capable of doing much more than when I first started last Fall!  This year, I hope to keep perfecting my skills and to learn new ones. I hope to continue to get a solid feel of what it's really like to work in a college lab before I head off to college myself next year!

Looking Back

Last October, I said that I was looking forward to my internship because I wanted to learn new things and experience a professional environment. After an amazing year, my internship has allowed me to do so much more than that. From the very beginning, I was integrated right into JP's lab and given as many hands-on jobs as possible. My only challenge was believing that I was capable of doing all JP thought I could! This year, I have had so much fun working with new equipment and processes in an environment that was not only professional, but encouraging for me to get involved. I have taken part in many different microarray experiments as well as smaller side projects, and I have had the opportunity to both set up experiments and analyze the data. Presenting my work to the school at the end of the year was an amazing opportunity for me to share my experiences with the Emma community. (Just to prove how supportive my mentors are, both JP and Dr. Karande came to Emma to see my presentation!) I would not trade my internship experience for anything, and I strongly encourage any Emma girl to do an internship if she is dedicated to the sciences and ready to experience what could come after Emma. My only suggestion for the program is to meet with the other interns more often so we can routinely share our experiences. My advice to future interns is don't be afraid to get involved. Everything may be overwhelming at first, but trust your mentors and you will have an amazing year! While it may sound like I am saying goodbye to my internship, I actually have the incredible opportunity to continue working in JP's lab over the summer. I thank everyone involved for an incredible year, and I can't wait to see what's next. Thank you for reading my blog! 

Presentation

Last Wednesday (May 1st), I presented the research from my internship to my school in our Student Achievement Assembly. I am so proud to be an example for all of the amazing work the Emma interns do, and presenting my work gave me a unique opportunity to expose the secret lives of the STEM girls. It was a great way to end a great year!

New Permeability Experiment!

This Tuesday (April 23rd), I went to RPI for another day to set up a new experiment! This experiment is similar in process to the experiment I was introduced to on December 11th. However, in that experiment, we were testing the natural permeability of the blood brain barrier, and this time we're testing the permeability of the blood brain barrier with our manufactured loop-2 protein added! As in the other experiment, the set up is two wells (one inside the other) with a filter on the bottom of the inner one separating the two, as seen in the figure below. On this filter, there is a monolayer of brain cells to represent the blood brain barrier.

We will have many of these setups in order to test solutions with different size molecules. We will be testing solutions with molecules that are 4 kDa, 10 kDa, 20 kDa, 40 kDa, and 70 kDa. All of these molecules are dextrans, or different size sugar molecules. For the normal type blood brain barrier that we tested earlier, the permeability to size curve looked like the black line below.With the peptide modulator that we have made, we hope to make the curve look like the red line below.

Today, we made the peptide solution to be added to the cell monolayer. We had to make sure that the protein was at a certain concentration in the PBS solution, so we used a Nanodrop spectrophotometer like the one below. The Nanodrop spectrophotometer analyzes the concentration of the solution by measuring absorption. 

We also changed the media of the cells to a non-phenol-red media. The cells are usually cultured using phenol-red media because it is a pH indicator. When the media is exhausted and needs to be changed, the media changes from purple to yellow. However, using this media in our experiment could alter the results. To change the media, we used a vacuum line to remove the old media and then added the new media to both the top and bottom wells. Once all of the materials were ready, we added the peptide solution and dextran solutions to the wells and started the experiment. 

I can't wait to see the results!

Graph Irregularities

This Tuesday (April 14th), I went to RPI to analyze data from another microarray affinity experiment. Affinity is the attraction to a substance, in this case a labeled protein. We used two different blocking buffers in this experiment. On one microarray, we used the BSA (bovine serum albumin) that we usually use with the microarrays. On the other microarray, we used a crude mixture (protein broth) from the company whose protein we are trying to purify, which contains contaminants as well as the protein of interest. Both of these microarrays were tested in concentrations from 1 nM to 3.5 nM. We graphed the data as intensity vs. concentration, where high intensity meant high affinity toward the printed peptide. The expected intensity vs. concentration graph would look like the graph below.

However, I looked through all of the graphs and found irregularities like the ones shown below. The third irregularity is especially strange because the intensity is very high at low concentrations, but very low at high concentrations.

After I finished recording the irregularities in the graphs, I looked at the kD values for the same experiment. I looked at each peptide sequence in relation to its position on the protein, and recorded the high and low kD values for each region.

Also, there was a vendor fair in our building today! We took a break to go look at the new technology, including movers, shakers, and much more. Overall, it was a lot of fun!

New Project

This Tuesday (April 9th), I finally went back to RPI to continue my research! JP introduced me to a new project that the lab is starting. Due to property concerns, I can't go into it in detail, but it has to do with a protein that binds to transferrin, which then attaches to a receptor on endothelial cells. This protein can then be moved through the cell into the brain via receptor mediated endocytosis. To produce this protein, we are going to use a cloning vector in E. Coli, which we will remove the protein from via osmotic shock. Each of the target proteins have a flag sequence attached. This flag will allow us to use an antibody that binds to the flag to purify the protein.

Our first project, explained earlier, is involved with opening a pathway between endothelial cells to allow transport into the brain. This new project also focuses on transport into the brain, but it is involved with making a pathway through endothelial cells.