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!

Amino Acid Solutions

This Tuesday (February 12th), I went to RPI for my weekly internship meeting. This week, I was in charge of making amino acid solutions to be used in the peptide printer. First, I had to calculate the volume of the solution that needed to be added to each amino acid powder. I was given each of the 20 amino acids in their powder form in separate tubes. The mg of powder in the tube was labeled on the side, along with the three letter abbreviation of the amino acid. I was also given a sheet of the dilution concentrations (mg and mL) for each amino acid. Using these numbers, I had to calculate the unknown, which was the volume of solution that needed to be added to the amino acid powder. For example, if the given concentration was 2 mg / 10 mL, and the tube contained 0.5 mg of powder, I would need to add 2.5 mL of solution. The concentrations for each amino acid were different, so I had to do that calculation 20 different times. After my calculations were finished, I went to the lab next door to use the fume hood to create the solutions. To do this, I used an Eppendorf that measured in microliters. The maximum volume that the Eppendorf could hold was 1000 microliters, or 1 milliliter, and the average volume I needed to add to the tubes was 4 mL, so I had to add the solution in smaller amounts. After each amino acid, I had to change the tip of the pipette to prevent contamination between amino acids. After adding the specified volume of solution to each of the 20 amino acids, I took the amino acid solutions back to our lab. There, I used a vortexer like the one shown below to dissolve the amino acid powder in the solution. Some were much easier to dissolve due to their polarity. These solutions will be used in the peptide printer to print arrays of peptides for future experiments. 

I'm looking forward to finding out what I will do next week!

Modeling Peptides

This Tuesday (February 5th), I went to RPI to continue my project from last week. Last week, I identified possible α helix binding sites on the protein. SInce last week, JP has worked on using the color coded model to come up with amino acid sequences that would form an α helix structure and bind to one of the sites. My job was to test the possible structure of the amino acid sequences by entering them into a program called PEP-FOLD. This program uses the amino acid sequence to identify possible  reactions between the amino acids that would make the protein's secondary structure. Ideally, the amino acid sequence we are looking for should have an almost perfect α helix secondary structure in order to bind well with the binding site on the protein. The figure below was the most successful α helix model we made. 

We also built the amino acid sequence using the MOE software. We set the program to make the sequence into the ideal α helix shape to determine where the bases would be located on the α helix. This allowed us to align our α helix sequence with its binding site on the protein model to identify the possible reaction sites and see how we could change the amino acid sequence to improve the binding. 

Overall, my work today really increased my knowledge about amino acid sequences and protein structure. I look forward to continuing to work on this project!

Protein Structure

Yesterday (January 29th), I went to RPI for more interesting analysis! We started with a summary of protein structure, which was really cool because we recently learned about protein synthesis and structure in AP Bio, so it was really cool to apply it to research! Protein structure is divided into different levels of structure. Primary structure (1˚) consists of the amino acid sequence of the protein. To form secondary structure (2˚), different amino acids that are not adjacent in the sequence can react and cause twists in the amino acid strand. The two common secondary structure forms are the α helix and the β pleated sheet. Today, as I will explain later, I focused on α helices. Tertiary structure (3˚) is formed when the secondary structure folds on itself, forming a three-dimensional structure. Finally, the quaternary structure (4˚) gives the protein form and function by adding molecules such as sugars and phosphates or combining different proteins together. Below is a diagram showing the different protein structures.

Source: Madison Technical College Lab Manual on Protein Structure

Today, my task was to look at a protein structure using MOE software. We are looking for a site to bind another molecule, especially the α helix sites. Two α helix forms can aggregate like a zipper, like the bindings of two spiral notebooks. I identified all of the different helix sites and recorded them by amino acid sequence numbers. After they were identified, I color coded them on the model so we could see where they were located in the three-dimensional surface structure of the protein. It was so interesting to see the relationship between the amino acid sequence and the final structure of the protein! It added a lot to my knowledge of protein structure and allowed me to apply something I learned in class.

As usual, I can’t wait to see what comes next!

Analyzing Data!

Today (January 22nd), I went to RPI for another day of interesting work! Today, I analyzed the data from the experiment I was introduced to on December 11th. To summarize the experiment, we were analyzing the size of particles that can normally pass throught the blood brain barrier, using a set up with two wells, one inside of the other (seen below). Twenty wells were tested. Ten had the dextrans added to the apical (inner) chamber, two each for the five different dextran solutions (4 kDa, 10 kDa, 20 kDa, 40 kDa, and 70 kDa). The other ten had the dextrans added to the basolateral (lower) chamber, two each for the five different dextran solutions. Further explanation of this experiment is given in my blog post from December 11th.



The data from this experiment came from solution samples taken at various times over a 25 hour time period. These samples were then scanned and analyzed according to their fluorescent signal. The dextran molecules contained Fluorescein Isothiocyanate (FITC), so the more fluorescnet signal the sample had, the greater the concentration of dextrans in that sample.

To analyze the data, I copied the data from the scan program into a program called Origin. There were three separate scans, so I had to average the data from the three. I then plotted the data for Concentration vs. kDa. After detecting errors in the beginning and end of the graphs, I trimmed the graphs to get a more accurate linear fit. After trimming the graphs, I calculated the r-squared values for each kDa. 4 kDa had by far the best fit. 10 kDa had the worst, and then the r-squared values increase for the 20, 40, and 70 kDa. After examining the Concentration vs. kDa data, I plotted the data for Concentration vs. Time for each of the 20 wells. For every well, the concentration increased over time. This is because as the time increases, more of the cell monolayer dies off, and more dextran molecules are allowed through the barrier. From the starting concetration values, it was evident that the larger molecules started with a lower permeability.

Overall, I had a very interesting day of analyzing data! I can't wait to see what's coming next week. 

Passaging and Protein Structure

Today (January 15th), I went to RPI for my first day of lab work of the new semester! Today was full of really interesting new things. First, JP showed me how to culture tissue, particularly the practice of passaging. Passaging is the process of moving some cells from a previous culture to a new growth medium to allow them to continue to grow. The first thing we had to do was spray everything down with ethanol. Everything must be sterile when working with the tissue, so all materials were sprayed, including our gloves. Next, we got the tissue culture out of the incubator, which maintains the CO₂ level and temperature similar to that of the human body. Before passaging the tissue, I was able to observe the culture under the microscope. The proteins that had been previously cultured were connected in a chicken pattern, and they were attached to the surface of the container with linkages that kept them in place. First, the media was removed from the container with a vacuum line. Then, 10 mL of PBS solution was added to the container to remove dead cells and remove the Fetal Bovine Serum (FBS) that inhibits the enzyme Trypsin from acting on the cells. Trypsin functions to cut the anchor linkages that connect the protein to the container, causing them to float around, which is not wanted while the tissue is being cultured. The PBS was then removed from the container with the vacuum line. Next, 1 mL of Trypsin was added to detach the cells from the container. After the Trypsin was active in the container for a few minutes, I observed the cells in the container again, and they were now floating islands of groups of cells. Next, FBS was added back into the container to re-inhibit the Trypsin. This mixture was then transferred to a conical vile to be spun down in the centrifuge. Once the centrifuge was balanced, it was spun at 1000 rpm for 5 minutes to make a protein pellet separate from the media. While the vile was being spun, we prepared a new container, and labeled it as passage #25 of the cells. 35 mL of new media were then added to the new container. After the vile was done in the centrifuge, the old media was drained from the vile using the vacuum line. We then added 1 mL of media to the vile to re-suspend the protein pellet by using the pipette up and down until the protein pellet broke up and dispersed in the media. 100 microLiters of the suspension were then added to the new container, and it was agitated to spread the cells around the surface of the container. The passage was then complete, and we re-cleaned everything with ethanol and put the new culture in the incubator.

After passaging, JP introduced me to a new computer program for modeling proteins. The program displayed both the amino acid sequence and the three-dimensional structure of the protein. My job was to search through the entire amino acid sequence to highlight certain amino acids that are important to the function of the enzyme. Once I highlighted the amino acids in the sequence, I also highlighted the corresponding structures on the enzyme that those amino acids code for. The purpose of this activity was to show the position of the binding site of the protein structure. After locating the binding site and seeing its structure, we looked at how the signal molecule (which is a sugar) binds to the binding site. This program was really interesting because I am learning about protein synthesis and structure in AP Biology right now!!!!

I can't wait to return next week and find out what's next!

Cleaning Up

12.18.12

On Tuesday December 18th, I went over to RPI for the last time before winter break. Because RPI was wrapping up their work as well, there were not any new experiments to be set up or new data to interpret. My day was full of cleaning and organizing the lab equipment and bench spaces. Also, I was able to see our lab's liquid nitrogen container, which was really cool! I can't wait to return after break and continue with our work.