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. 

PBS Solution!

12.11.12

Last Tuesday (December 11th), I went over to RPI for another day of lab work! Today's work was preparing for a new experiment that is analyzing the size of particles that can normally pass through the blood brain barrier. As seen in the figure below, the set up for this experiment will be two wells (one inside the other) with a filter on the bottom of the inner one separating the two. On this filter, we will grow 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, the permeability to size curve would look like the black line below. With the peptide modulator we are currently researching to find, we hope to make the curve look like the red line below.

In preparation, I prepared two Liters of phosphate buffered saline solution. This PBS can be used to dialyze protein solutions. To make this PBS, I combined 15.52 mL of 1M K₂HPO₄, 4.48 mL of 1M KH₂PO₄, 2 L of distilled water, and 11.6 g of NaCl. After mixing these contents, I had to raise the pH to 7.4 using 1M NaOH base. After many trials and test with a pH probe, the pH reached 7.4, and my PBS was ready to be filtered. To filter the solution, I used a bottle-top filter with a vacuum attached, like the one shown below.

My second job was to label the test tubes for the experiment. 10 mL samples will be taken from the bottom chamber 12 times over a 9 hour period. I labeled the test tubes needed for the first 5 data collections by writing the time on the cap and the time- kDa of the solution on the side of each tiny test tube.

I can't wait until next week to hear the results of the experiment!

Lab Work!

12.4.12

This Tuesday (December 4th), I went over to RPI for my first day working in the actual lab. The goal for today was to print 15 slides of peptide matrices to be able to run tests on in the future. There are 56 peptides that we are working with, and each peptide is printed in a 3X3 matrix on each slide. This means that there are 9 dots per peptide, and 504 dots per slide! Ben showed me how the computer program worked, and we checked the (x, y, z) coordinates of the printing needle on each slide so the machine would run correctly. The x and y values were mostly already correct from previous runs, but the z coordinates (height) needed some adjustment so that the needle was touching the slide. This placement of the needle allows the machine to dispense the correct amount of peptide on the slide. The printer works automatically after the programming is set, and the whole process takes about 3 hours! After double checking the coordinates, Ben showed me how to use the centrifuge to spin down the peptides. When the peptides were ready, we put them in the printer, and started the program. We had to watch it through its first cycle to make sure that it wasn't missing any dots because the machine often doesn't function correctly. Today the machine was running well, and we did not see any malfunctions!

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

Training

11.28.12

This Wednesday (November 28th) due to the school schedule change, I took my
trip over to RPI. After arriving late due to transportation confusion, I promptly
began my task for the day. I had to complete two training courses before I could
participate in actual lab work. This training consisted of about two hours
containing courses in both general lab safety and biohazard safety. The training
used an online course that consisted of text slides, audio, and video, and "tests"
were included along the way. After learning a ton about MSDS (material safety
data sheets) and different safety controls in the labs, I passed the final exam
(especially because the program made you retake it until you got a perfect score!).
After my training, JP took me to get my official RPI ID card. After giving many
forms of legal identification, the clerk took my picture and made me an official
part of the RPI system! I can now enter the building, office, and lab by
myself (finally!)! 

My ID!

Next week, I am hopefully going to participate in the lab because I have now 

completed all of my training. I can't wait!