Hello everyone!! I’m Azari and I just completed my fourth week at Scripps Research. Just a refresher, I’m working with Nate Beutler, a post-doctorate immunologist who, through antibody discovery and therapeutic design, is working to understand why an effective vaccine for malaria hasn’t been developed yet. This week I preformed an ELISA (enzyme-linked immunosorbent assay), started a new type of cell culture, and spent lots of time at the beach!
Prepping the ELISA
ELISA is a test to measure the amount of antibodies there are in a sample. We tested how many antibodies there were in our transfections from last week. We used samples from day 0, day 1, day 2, day 5, and our standard (control) to see which days produce the most amount of antibodies. Yenni also had us test one of his samples. The ELISA works by attaching a primary to the inside of tiny wells. The well plate is stored overnight and washed in the morning. The wells are then coated and incubated with BSA (bovine serum albumin), a protein used to bind molecules. We then added our antibody and let it sit for about 45 minutes. The well plate is then washed and we added our substrate (the molecule that glows for visualization). I used alkaline phosphatase because it binds the best with my antibody, mAb311. We did a dilution gradient so our first row was our antibody neat. Our dilution factor was 5 so I took 1/5th of the first row and added it to the next row and so on. So, the first row was the most concentrated with our antibody and the last row was the least concentrated. We analyzed our results with a spectrometer and everything looked good. My ELISA definitely needed to be incubated more, it wasn’t as visible as Shiloh’s. It’s not a big deal though, it was expected that mine would be a bit less vibrant.
Pulling the cancer cells from the freezer
Nate B. also had us start a new kind of cell culture on Monday. There’s two kinds: adherent and suspended. Our Expi293 suspended cells are used for protein expression while new adherent cells for different assays (like neutralization assays). First, we made our media. It’s made up of FBS (fetal bovine serum, helps the cells stick), pen/strep, and L-glutamine. While we warmed the media, Nate took us to the basement to get our cancer cells from a huge freezer. My cells were Vero monkey cancer cells which are used for pathogens such as SARS-2, Zika virus, and dengue. Shiloh chose a type of cancer cell used for malaria (she stole them from me). These cancer cells will grow forever as long as the have nutrients, unlike our Expi293 cells. My cells grow a bit slower than Shiloh’s so I didn’t have to split when she did. Splitting is a challenge in itself since theres no machine to tell us how confluent our cells are. We have to estimate when our cells are about 80% confluent and split then. Mine were at about 50% on Thursday so I left them to grow over the weekend. Also, I named my cells ‘A$AP Rocky’ (because my other cells are named Rhianna) and Shiloh named hers ‘Biggie Smalls’ (they were in a tiny flask and she thought it was funny).
Wednesday was lab clean up day so we spent the morning cleaning the centrifuges. According to Nate B. and Sean, this is the easiest task but it still took Shiloh and I about 45 minutes. We helped with some restocking and we also helped Nate B. clean an incubator full of measles (don’t worry I’m vaccinated!!). This took up a pretty large portion of the day. With not much time to start another project, Nate L. had us complete his gigapreps in under 2 1/2 hours. He needed them done to be sent for sequencing by 5pm, but unfortunately we didn’t finish by then. When we finished he admitted he wasn’t in any rush to get them sequenced and that he really just wanted to see how we worked under pressure. Aside from the time crunch I’d say we were a really good team! He counted them and our count was very high. We finished our day with Nate B. as he showed us how to split our adherent cells.
Plasmodium falciparum undergoing confocal fluorescent microscopy
Thursday was definitely my favorite day this week. We started our morning with calculating and splitting our suspended cells for them to be at 3 million on Monday. Mine were a bit overgrown and the viability wasn’t great so hopefully they’re okay. We did an SDM (site-directed mutagenesis) with Sean while Nate B. figured out some stuff for work. Site-directed mutagenesis is a technique to study proteins by introducing a certain mutation into a DNA sequence. After finishing up Nate B. took us to another building to observe his sample of Plasmodium falciparum through confocal fluorescent microscopy. This has to be one of the coolest things I’ve got to experience so far. The parasite is stained with green florescent protein and a laser is pointed at the parasite to excite it. This specific sample wasn’t working as well as Nate wanted it to but it was still cool to see. I really appreciate that although he was clearly upset about the sample not working, he still kept a smile on his face and played relaxing music. I’m starting to really see that science is all about making mistakes and not letting them discourage you. We returned to the Immunology and Microbiology building and he explained our project for next week. We also prepared a protein gel but weren’t able to see the results because we were in a rush to go surfing with Nate, his girlfriend Marie, and Yenni. They were all insanely good meanwhile I got a cut in my face from the fin because I fell wrong (it’s not as bad as it sounds) and Shiloh tripped while running with her surfboard in hand while trying to chase a seagull. Although a lot went wrong, it was so fun that were going again in the morning. This time I won’t fall under my board!! We had dinner and crepes with them. Thanks Nate and Marie!
Group photo on the 4th!
Friday was July 4th so of course we went to the beach for majority of the day. We tanned while listening to music, made friendship bracelets, went boogie boarding, and built a sandcastle. I also spent too much money at Wetzel’s Pretzels. We returned home, watched the newest episode of Love Island, grilled some burgers, made s’mores, and watched the fireworks from our backyard. Saturday was very laid back (much needed). We headed over to a nearby mall where Lana and Maiya found some cool clothes and jewelry. Shiloh and I found some evil eye charms that resemble our cells in trypan blue so we got them. I can’t wait to start a new week but it’s starting to hit that its almost time to go back home. I plan on making the most out of my last two weeks here both in and out of the lab.
Thanks for reading, see you next week : )
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