Concentration Experiment!

Today (February 19th), I went over to RPI to continue my work in the lab. Since last week, slides of peptide arrays were printed using the amino acid solutions I made. Today, I helped use these printed slides to set up and run an experiment. In this experiment, the goal is to determine what part of loop 1 of the Claudin-5 protein in the endothelial cell tight junctions reacts with loop 2 on the opposite side of the junction. These loops can be seen in the diagram below. 

In this experiment, we are using a solution of 90% unlabeled peptide and 10% TAMRA labeled peptide. The TAMRA binds to terminal alkynes via a copper-catalyzed click reaction, and it is fluorescent red. The fluorescent tagging is useful in determining where the peptide binds to the printed 56 peptide array via intensity analysis. In this experiment, There are 10 slides containing 56 printed peptides each. Each of the slides will be exposed to different concentrations of TAMRA tagged peptide, the highest being 500 mM and the lowest being 100 mM. On the highest concentration slide, we expect to see all of the dots tagged at a high intensity, and on the lowest concentration slide, we expect the TAMRA tagged peptides to bind only to the highest binding proteins on the slide. To analyze the intensity of each peptide binding spot, it will be scanned into a computer program, and the intensity will be evaluated using pixels. This data will then be plotted on an intensity vs. concentration graph. We expect the data to resemble the graph shown below, with the intensity increasing as the concentration increases and eventually plateauing. This graph will then be used to find 1/2 of the plateau intensity, and identify the concentration of that intensity. This value will be used to find the Kd, or the affinity of the peptide towards the substrate. 

In my work today, I helped to set up this experiment. Overnight, the slides were placed in individual petri dishes and coated with BSA (Bovine Serum Albumin) to prevent the peptide from binding with the blank space on the slide where no peptide dots are printed. My first job was to wash the excess BSA off of the slide with PBS. The PBS solution I used today was actually the PBS solution I made on one of my first visits to the lab! To wash a slide, I first poured the excess BSA out of the petri dish. Then I added 10 mL of PBS to the petri dish, making sure to not add it directly onto the slide to avoid forcefully removing BSA from the slide. I then repeated this process for the other nine slides, and then put on them on a rotator for 10 minutes. This process was repeated three different times to ensure that all of the excess BSA was removed. 

While the slides were on the rotator, JP and I did calculations to determine the volume of unlabeled peptide, TAMRA peptide, and PBS solution to put on each slide. The amounts were different for each slide because the concentration of TAMRA peptide was different. To add the peptide solution, we used the properties of surface tension, as in adding water droplets on a penny. However, the slide had to remain wet, so it made the process more difficult. First, I poured the PBS solution off of the slide. Then, JP dried the sides and bottom of the slide to allow us to use the properties of surface tension. While JP was drying the slide, I dried the petri dish with Kimwipes. The slide was then put back in its labeled petri dish. We then added the specified concentration peptide solution to the slide, making sure it was evenly dispersed and that none went over the edge of the slide. We then repeated this for each of the slides, using the different concentration peptide solutions. After all of the slides were coated with their specified solutions, we covered them with black paper because the TAMRA is light sensitive. The slides are going to sit and be slowly rotated for three hours. 

I can't wait to see the results from this experiment next week! It should give us useful information going forward in our research. It was very interesting today to see different activities I have done in the lab come together. The amino acid solutions I made last week were used in the printing of the peptide slides, and the PBS solution I used today was the solution I made! I look forward to continuing my research and learning more new procedures!