Farewell to My Friend
Table of Contents
Sunday, March 2
165.0 lbs (Day 65, starting from 162.6 lbs on Dec 28) | 🏃🏻 Done (6/6) | ★★★★☆
After the usual morning workout with my friend, we had brunch with a few Berkeley PhD students that my friend knows, and since the weather was nice, we headed to a park near the Palace of Fine Arts (3601 Lyon St, San Francisco, CA 94123). The wind was quite strong, but the sun was shining, and it reminded me once again that I should make it a point to get out somewhere at least once a week. Everyone I met was really friendly, and we agreed to go hiking together next time if we get the chance!
After that, I finished catching up on my blog posts at home, completed an assignment that’s due tomorrow, and then went to sleep. Oh, and the whiteboard finally arrived!! I’m planning to barricade my bed with it this evening haha…. If all this effort still doesn’t work out, then I honestly won’t know what to do. I believe it’ll be a success🥹
Monday, March 3
164.6 lbs (Day 66, starting from 162.6 lbs on Dec 28) | 🏃🏻 Rest (0/6) | ★★★★
Outside of Work
This morning, I skipped my gym session because I had to visit my primary care doctor for my second dose of the HPV vaccine. After getting the shot, I felt a little sore, but overall, the day went well.
Today was a busy day because I had two classes. One was a regular reaction mechanism class from 1:00 pm to 2:30 pm. In addition, once a month we have another RCR class, and today was that day. After classes, I headed home quickly, had a late lunch, and then returned to the lab. I finished my experiment around 10:30 pm, so I didn’t have time for any other activities at home.
Lab Work
Biology:
- M→C: I began by thawing the pellet I had purified last Saturday, followed by a Nickel Column, Overnight TEV Cleavage, and Buffer Exchange. It went so well. I believe it was because it was my third attempt👍
- E→C: I made a starter culture for protein expression by inoculating a colony from the plate I had stored in a 4°C freezer.
Tuesday, March 4
165.4 lbs (Day 67, starting from 162.6 lbs on Dec 28) | 🏃🏻 Done (1/6) | ★★★★
Outside of Work
I went to the gym and did a push workout. I had a class scheduled but I can miss up to three classes each quarter. So I decided to skip it because my schedule was full of experimental work, so I had to set my priorities.
After work, I felt like cutting my hair, so I gave myself a haircut, probably the tenth time I had done it. The last time did not go well because I cut the top of my hair too short and ended up wearing a hat for two weeks🥲, which was embarrassing. Today, I chose a relatively short style, and when I looked in the mirror, it looked pretty good. After that, I cleaned my house and went straight to bed.
Lab Work
Biology:
- M→C:
- Yesterday, I performed an overnight TEV cleavage and buffer exchange. This morning, I checked the results using LC-MS and SDS-PAGE. Both tests showed that the cleavage was complete and that the target protein was present.
- I moved on to the next purification step using reversed nickel NTA affinity chromatography. The LC-MS results at this stage were good. I then concentrated my sample from 60 ml to 1 ml in preparation for size exclusion chromatography. After the concentration of the sample, LC-MS confirmed that it was pure, which was great.
- Later that night, however, I ran into a problem. When I used the nanodrop to measure the concentration of my purified protein sample after SEC, I noticed that the concentration was too low and the absorbance pattern looked odd. After some troubleshooting, I found that the absorbance of the sample right after SEC (before concentration) was much higher! than that of the concentrated sample. I then checked the filtrate, and LC-MS detected my protein, although it showed a low absorbance. This means that the issue was with the centrifugal filter I used.
- E→C: overnight 18oC protein expression (2 x 1L TB)
Wednesday, March 5
165.8 lbs (Day 68, starting from 162.6 lbs on Dec 28) | 🏃🏻 Done (2/6) | ★★★★
Outside of Work
I started my day with a refreshing morning workout. Afternoon, I attended a reaction mechanism class. I spent the rest of the day on experiments(+ group meeting). These experiments, conducted from Monday through Wednesday, have been quite intensive, and I ended up working until 9 or 10 PM. It has been exhausting, but I’m pleased with the progress.
Lab Work
Biology:
- M→C:
- Yesterday, I identified an issue with the membrane filter used in a centrifugal filtration process to concentrate the protein sample. With the PES membrane (10K molecular weight cutoff), I found that my protein was present in the filtrate, and the concentration in the retentate was significantly lower than expected. To address this, I decided to use a membrane made of a different material that has a smaller molecular weight cutoff. Today, I switched to a regenerated cellulose membrane.
- According to a manual, the cutoff should be at least three to six times lower than the molecular weight of the protein of interest. Since my protein is approximately 24K, I needed a cutoff between 4K and 8K. A 10K cutoff was, therefore, too high, so I switched to a 3K cutoff. This time, I ensured that the concentrated sample was resuspended every 10 minutes instead of every 20 minutes and used a spin basket rather than a fixed-angle rotor.
- Fortunately, after filtration with the 3K membrane, I did not detect any protein in the filtrate, and the retentate showed a higher protein concentration compared to the initial attempt with the 10K membrane. Overall, I successfully recovered approximately 2.2 mL of concentrated sample. I plan to run size exclusion chromatography on this sample tomorrow.
- E→C:
- Yesterday, I set up overnight cultures for protein expression. I followed the standard purification protocol: first, I harvested the cell pellet from the cultures, then added lysis buffer, performed sonication, and centrifuged the mixture to obtain the lysate. Next, I carried out nickel-NTA chromatography.
- The SDS-PAGE results were surprising; I found my protein even during the wash step, which is designed to remove proteins that do not bind to the nickel resin. Despite this, LC-MS analysis confirmed that the protein was relatively pure. I also verified the composition of other fractions, and the results were good. I moved on to dialysis and simultaneous TEV cleavage via buffer exchange in the cold room.
Thursday, March 6
166.0 lbs (Day 69, starting from 162.6 lbs on Dec 28) | 🏃🏻 Done (3/6) | ★★★★
Outside of Work
Hit the gym, no classes… only focusing on experiments. Working until 10 PM for four days straight feels a bit exhausting. It’s a relief that the results are finally improving. It’s really tough that there’s not much time left in my rotation, and I absolutely have to get the data😇
Lab Work
Biology:
- M→C: Finally after running SEC, I got enough protein for labeling experiments(and nanodrop is fine). Next time I will make sure the cutoff of a centrifugal filter is optimal😭
- E→C: TEV cleavage was good, but I barely found my protein after reversed-Ni, even though I pushed the imidazole gradient high. I had half of the cleaved sample, and next time, I will try again and use the sample before the reversed Nickel as a control in SDS-PAGE to see if the reversed Nickel column is the actual problem.
Friday, March 7
165.6 lbs (Day 70, starting from 162.6 lbs on Dec 28) | 🏃🏻 Done (4/6) | ★★★★
Outside of Work
Today was actually not much time to do experiments. This morning, I attended a class and then joined my friends in a protest against the current government’s NIH funding cut policy. I thought that, as an international student, I wouldn’t be directly affected, but the situation turned out to be more serious than expected. It might even impact my Fulbright scholarship. Moreover, many labs at UCSF depend on NIH funding (cuz most focus on translational research), so there’s a pervasive sense of concern. In fact, some universities are even drastically reducing their graduate admissions this year… I really hope this is resolved soon. After the protest, I attended an RCR discussion class and did some experiments in the lab, and for the first time in a while, I left work before 8 PM!
Lab Work
When I arrived this morning, I discovered that someone had left the precious compounds I had synthesized so far at room temperature… They should have been stored at -20°C🫤. Fortunately, the graduate students and postdocs assured me that it probably wasn’t a problem (the compounds aren’t that unstable), but I still couldn’t shake off the unpleasant feeling.
Biology:
- E→C: I collected all fractions identified by LC-MS as containing my protein and ran SEC. LC-MS was fine, but the protein concentration was too low.
Saturday, March 8
165.4 lbs (Day 71, starting from 162.6 lbs on Dec 28) | 🏃🏻 Done (5/6) | ★★★★☆
After a long time, I met with some friends at Stanford who attended the same pharmacy school in Korea, as well as a friend who was a year behind me in pharmacy school and successfully went through this year’s graduate school admissions process, coming to Stanford for an interview. I also met someone who recently started as a freshman in Stanford Immunology. Since my friend had just finished her interview and needed to return to Boston, we grabbed a meal and coffee near the airport at a Hawaiian restaurant (specializing in ube, a type of purple yam) and chatted about various topics.
In fact, one of my dear friends who helped me so diligently with my own admissions process is temporarily returning to Korea, which left me feeling a bit empty. I once hoped we could continue to stay together, but I suppose it’s best to support his future plans. He should try everything he wants and maybe return depending on the circumstances. In any case, I hope he pursues all his plans without regrets. I really support him!
After seeing off my friend from pharmacy school at the airport, a friend took me to the UCSF library. I worked on my reaction mechanism and assignments, and now that I need to start my labeling experiment, I did some research on it. I also wanted to try out a structure prediction model called Chai-1, so I looked into the school’s computation cluster, Wynton.
