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.

Microarray Model

Last Tuesday (April 2nd), my mentor was out of town, so I did not go to RPI. However, my mentor gave me a project to work on for the week! We are working on taking concepts involved in our research and turning them into hands-on craft projects for children in grade school through high school. My project is to come up with a craft demonstration of how a microarray works. A microarray is a glass slide that usually contains nucleotide or amino acid probes that bind certain molecules. My concept is to use Velcro to demonstrate the binding of the printed peptide propes to molecules in the solution the microarray is exposed to. In our research, we use amino acid probes to make a microarray that consists of  56 peptides, each in a 3 by 3 matrix. For my demonstration, I am planning to use cardboard to represent the glass slide of the microarray. For the peptide probes, I am planning to use adhesive circles of Velcro. Some of the Velcro circles will be the hook side, and some will be the loop side. For the binding molecules, I will use Styrofoam spheres that have one kind of Velcro, hook Velcro for example, attached. These "molecules" will then bind to some of the "peptide probes," but not to others. To simulate the coating of the microarray, the "slide" will be put in a box, or "petri dish," and the "molecules" will be poured in. The box will then be moved in circular motions like it is on a rotator. After being "coated," the excess balls will be pored out of the box, and only the bound "molecules" remain attached to the microarray.

We also discussed having a second part to the activity using a "mixture" with varying sizes of molecules to demonstrate epitope mapping. Overall, we hope to use this demonstration to aid learning about microarrays, protein interfaces, purification of solutions, and peptide engineering.