Week of 11/28/16

This week, Yi and I a pretty hands-on lab day. We performed out mini experiment involving different solutions of protein, urea, and buffer to measure how concentration of urea affects protein folding. After combining a solution of buffer and protein with another solution of buffer and urea, we used advanced spectroscopy to measure the intensity of proteins in the folded versus unfolded proteins at different concentrations.

I had to make a table of expected molarities, prepare the two different solutions, and combine the solutions in the appropriate ratios to perform the spectroscopy. Making the table of expected molarities was pretty simple, as Yi told me what the total solution volume should be (120 microliters) and what the expected urea concentration was. From there, all it took were a few molar conversions to figure out how many microliters of each solution were necessary. Below I have recorded the table of expected molarities.

Concentration (M)	Volume of protein + urea (µL)	Volume of protein + buffer (µL)
0	0	120
.2	6	114
.5	15	105
.5	24	96
1	30	90
1.2	36	84
1.4	42	78
1.7	51	69
2	60	60
2.3	69	51
2.5	75	45
2.7	81	39
3	90	30
3.3	99	21
3.6	108	12
4	120	0

Once the table was finished, we had to prepare the solutions. I used a scale to measure the proper amounts of urea to add to the protein solution, using molar mass to determine the number of grams of urea necessary for creating the proper concentration of solution. We then mixed the predetermined volumes of urea/protein solution with the buffer/protein solution to make the necessary concentration in twenty different vials. It should be noted that the amount of protein was constant in each vial, because our independent variable was urea concentration, not protein concentration.

We then place the vials of varying solution into the spectrometer to get a measure of protein absorbance under different urea concentrations (and thus proteins in different folding states). It took awhile for us to get results for our mini experiment because the spectrometer had to be warmed up and calibrated for the light intensity we were using. Unfortunately, I could not download and print the results of our experiment, but I can say that the peak absorbance lay within the middle range of urea concentration, after the pH was too basic but before it became too acidic and the protein denatured.

Although our experiment was brief and not very official,it was great practice in the lab and gave a good overview of the equipment and techniques we will be using in the future!

Week of 11/14/16

This was my first week back since October. As explained earlier, I was in New York City a few weeks ago on an opera trip, and last week I had to attend an unexpected funeral. Needless to say, I was very excited to get back to work at RPI. Although my visit this week did not go as expected, it was enjoyable nonetheless.

Yi had a big presentation to give after our visit on Tuesday, so we didn't have enough time to visit the lab this week. Instead, we sat in his office and he walked me through his PowerPoint, which he will present at a large conference in New Orleans in a short time. Before you speculate that this week was wasted, I will assure you that his presentation was very much related to what we are working on.

In his experiment, Yi used HSQC (heteronuclear single quantum coherence spectroscopy) and high pressure NMR (nuclear magnetic resonance spectroscopy) to investigate the relationship between pressure and protein states. Essentially, he was looking at the behavior of folding proteins under different pressures. Do more proteins enter the folded state under really high or really low pressures? Generally, Yi found the answer to be somewhere in the middle. Moderate pressures tended to work best for proteins in order for them to fold and take shape. Pressures that were too high or too low seemed to cause the folded proteins to denature and remain in the unfolded state.

I do not have a picture of Yi's presentation, but I have pictured below a sample NMR graph as well as a few images that illustrate that ideal pressures for protein folding lie not on the minimum or maximum extrema, but within the middle range of the pressure values.The wavelength with the highest intensity of folded proteins correspond to mid-range pressures.

NMR intensity image. Source: http://www.cbs.cnrs.fr/index.php/en/research-equipe1/rmn-haute-pression

This week did not go as expected, but it was really fun! I really appreciated Yi's willingness to share his presentation with me. He will do great in New Oreleans. Until next time!

Week of 10/31/16

I was in New York City with the choir this past Tuesday. We got to see an amazing opera, and I had so much fun. Unfortunately, this meant that I could not attend my internship. Because we did not meet, I decided to take a few minutes once we returned from the trip to gather resources for my amino acid studies. The most useful diagram I found is shown below.

Amino acid study chart. Source: https://en.wikipedia.org/wiki/Proteinogenic_amino_acid#/media/File:Molecular_structures_of_the_21_proteinogenic_amino_acids.svg

I also love Leah4sciMCAT's videos.She has clear, concise presentations on amino acid structure, function, and properties. Here is a sample video about basic structures of amino acids.

Amino acids introduction to structure charge classification and reactions video.
Source: youtube.com/leah4scimcat

All of these resources make it so much easier to study amino acids. Thank goodness for modern technology! Can't wait to get back to my internship next week.

Week of 10/24/16

This week was fun! Yi didn't have any specific plans in the lab today, so we made plans for a mini experiment that involves measuring protein fluorescence (and by extension, structure) when the protein is exposed to different levels of urea. Urea is an organic compound that plays important role in the metabolism of nitrogen-containing compounds. It is used in many bodily processes, particularly involving nitrogen excretion.

Before beginning our work, we had to isolate a protein sample from a protein-buffer sample. To do so, we used a centrifuge. A centrifuge is a machine with a rapidly rotating container that applies centrifugal force to its contents, typically to separate fluids of different densities. When the centrifuge is finished its cycle, the protein and buffer solution separate, leaving an isolated layer of protein that can be in an experiment. A detailed picture of how this machine works is below.

Centrifuge diagram. Source: http://cdn4.explainthatstuff.com/centrifuge-high-speed.png

Once we had our protein sample isolated, we had to make a plan as to how we would alter the concentration of urea in each sample we will use to examine protein fluorescence. The ratio of urea to buffer needs to increase each time. But first, we needed to make the stock concentration of urea to be diluted. We made a solution of buffer and urea, and I had to do all the molar conversions and manipulations to translation the number of grams per milliliter of each substance.

Once the calculations were done, we massed out the number of grams of urea and CC necessary and combined them in the buffer solution. Urea takes a very long time to disolve in water, so we placed our mixture in a warm water bath and shook the combination jar very vigorously. By the time the urea had started dissolving, our time was up! Yi had a chemistry lecture to get to and I had a transport van to catch. This week was a fun one, and I am so excited to go back. Until next time!

Week of 10/16/16

This past Tuesday, I visited RPI for my first lab session! I received my own official badge that allows me access into the CBIS building and laboratories. This was a cause for great celebration for both me and Yi, the graduate student I am working with (who no longer has to race to meet me at various entrances when I call to be allowed entry into the building).

Besides receiving m own visitor's badge (and a pretty rad pair of safety goggles), Tuesday was the first day of official (ish) lab work for me. Yi took me around the lab, giving me a tour of his designated station and all the equipment we will be using throughout this process. I discovered the centrifuge, uber-fancy pipettes, protein fridge, and more. Once I was familiarized the area and debriefed on laboratory safety, we began working.

Since it was my first official day, Yi wanted to make sure I understood the basics of lab techniques we will be regularly employing during our work this year. We talked about calculating protein concentration using absorbance, the molar absorptivity constant, and path length (hello, Beer's Law!). Essentially, calculating the protein concentration can be boiled down to dividing the absorbance by the absorptivity coefficient (m.a. constant x path length). I was really happy to discover that I already knew and understood what Yi was explaining to me before he finished, and his recap of Beer's Law was a great refresher. We were both (at least, I think it was both of us!) pleased to realize that my science background had prepared me well for this experience, and it was time for lab work to begin.

I participated in a simple spectroscopy demonstration under Yi's supervision. We took a small amount of DI water and placed it onto the slit of the spectrophotometer, Then, we closed the machine and allowed it to run. This set the blank for the machine. Next, we loaded a small amount of our protein sample onto the slit, and allowed the machine to run. Eventually, the computer relayed the result of the concentration of our protein sample. How does a spectrophotometer work, you ask? Here is a simple picture that explains, though ours did not occur within a cuvette.

Spectrophotometer diagram. Source: http://chem.libretexts.org/Core/Physical_and_Theoretical_Chemistry/Kinetics/Reaction_Rates/Experimental_Determination_of_Kinetcs/Spectrophotometry

After the lab seesion was done, I went back to Yi's office and watched videos about amino acids. I need to be easily familiar with the different groups- knowing their structures, properties, and names will be crucial for this experiment. I've continued my exploration of amino acids this past week, and hope that I will be a pro on all the R groups this time next week. Wish me luck studying!

Week of 10/09/16

My first post! This year I am taking part in the CBIS High School Scholars Program at Rensselaer Polytechnic Institute. I will be interning under Dr. Catherine Royer at the Center for Biological and Interdisciplinary Sciences (CBIS) at RPI, and working on protein purification. Protein purification is a series of processes intended to isolate one or a few proteins from a complex mixture, usually cells, tissues, or whole organisms. This process is necessary for characterizing the structure, nature, and interactions of a selected protein.

Protein purification flowchart. Source: http://2009.igem.org/Team:Washington/Project

This past Tuesday our introductory meeting took place. I met with Mr. Calos, Dr. Royer, and Yi, the graduate student I will be working with, to discuss the meeting times a procedures for this year-long internship. We discussed my background in science, how laboratory proceedings will occur, and the expectations set for me as my senior year (and this internship) continues. I found out very quickly that I really enjoy the company of both Dr. Royer and Yi, which only makes me want to succeed even more. Hopefully, I will be able to put in enough time, effort, and enthusiasm to make this internship a success. Until next time!