Internship Log

Week 1 (5/21/18 - 5/25/18):

• Monday: My first day at NIH (in 2018, at least)! I have previously worked in the NICHD (Eunice Kennedy Shriver National Institute of Child Health and Human Development) as a rising senior in high school. My first time at NIH, I was mostly doing shadowing (clinical rounds, surgeries, and lab work - oh my!), so I’m excited to see the “other side” of what NIH has to offer. My first day was relatively uneventful, as I was the first intern to arrive on campus (not only in my lab, but in the entire NOB branch). I got a desk and computer, and was mostly wading through the boatloads of paperwork and trainings that NIH requires interns to complete. I met Adam upon arriving, the overall lab manager for the ~5 labs in my branch, who led me to my lab and introduced me to Herui, who I believe will be my mentor. Herui is quite busy (at all times), and spent a lot of time in the “animal facility” - not much more detail was given than that, but I hope that I can go with him next time to learn as much as I can about my lab’s research.
• Tuesday: Unfortunately I had a sick day today! Woke up feeling awful, so I called Herui, who said it was fine, and that we could discuss my research project when I get back.
• Wednesday: I learned today that not only am I the first research intern to arrive on campus, but that my Principal Investigator (PI), Dr. Zhengping Zhuang, M.D. Ph.D. is at an international conference, and will not be back in the lab until next Monday. Herui and I agreed that we should wait until Ping (Dr. Zhuang) gets back, so we can agree on a project that fits my own research goals, and will help the lab reach its goals, too. Since I have had very limited research experience before this summer, I have been spending a lot of time shadowing Herui, and learning the ins and outs of lab. I also met Qi and Jing, two wonderful ladies who are postdocs in my lab, working on research under Ping. I learned that each of them will be taking at least one summer intern, all of whom are arriving in the next 2-3 weeks.
• Thursday: I have been shadowing Herui for most of the day today, again! Although I wish I could already start my research, it looks like I will have to wait until next week to really solidify my plan. I made a goal today to read at least 1-2 scientific papers (relevant to my lab’s research) each week in lab (while waiting for experiments), so I can become well-versed in our studies, as well as terminology and lab techniques.
• Friday: Only really did online training today - after I set up my NIH email and got it confirmed, my flooded inbox notified me that I needed to complete official online trainings before conducting any research (and before a set deadline!). I watched Herui transfect cells today, and he explained a lot to me about the HIF-2A hypoxia signaling pathway. We did some imaging of cells transfected with GFP, targeted to HIF in the cells, and I went with Herui to the lab in building 37, where they have a special microscope-imaging system and software. In the dark room, Herui showed me how to do the imaging, and let me do it myself - I took several labeled images for Herui’s research and transferred them to a flash drive for later use.

Week 2 (5/28/18 - 6/1/18)

• Monday: Memorial Day! Federal Holiday; NIH is closed.
• Tuesday: Things really started to pick up today! A second summer intern arrived, who I spoke to for a long time. Her name is Francia, and she is a rising Senior at Duke University. Her family lives in the area, so this is actually her third summer completing the Summer Internship Program at NIH with Ping’s lab. She told me about the different work she’s done the past few summers (last summer, she mostly did cell work), and is now planning on working on cells with an IDH1 (isocitrate dehydrogenase 1) mutation. The cellular system they are planning on using for her experiments is called iPSC, which stands for induced pluripotent stem cells (which are obtained from actual patients with neural system tumors here at NIH). Francia also discussed with me what she thought my project might be, and explained (in detail) the differences between Normoxia and Hypoxia, and how this affects HIF.
• Wednesday: After talking with Ping, Francia, and Herui (who is also serving as Francia’s mentor this summer), we decided on a preliminary plan for my experiments, which can be tailored once we figure out how long each portion will take. In summary, my project will consist of analysis of two key proteins in the hypoxia signaling pathway - VHL (Von Hippel-Lindau) and HIF-2A (Hypoxia-Inducible Factor 2A). Our studies will probe different mutations in each protein, and analyze the downstream effects of these novel mutations. Specifically, I will analyze the VHL protein with a mutation at amino acid 54, a point mutation from Methionine to Isoleucine (M54I). Additionally, I will do two separate assays with the HIF 2A mutated protein (E548K - Glutamic Acid to Lysine at the 548th amino acid) - an assay with CHX, which is a protein synthesis inhibitor, to test for the point in the cell cycle at which the HIF 2A mutation affects the hypoxia pathway and the protein half-life, as well as a Ubiquitination assay to test the level at which the mutated protein is ubiquitinated (tagged for the Proteasome and signalled for subsequent destruction). I continued my training with Herui after our meeting.
• Thursday: For the VHL mutant (M54I) our model system will be E.Coli, and we will transform a bacterial plasmid vector into the competent cells. Today, we extracted the plasmid DNA from cultured colonies in order to determine if the DNA was taken up and expressed correctly - we sent it (via a dropbox in our building at NIH) for sequencing overnight. If the vector is verified come tomorrow morning, we will expand it with polymerase chain reaction (PCR). We used a kit to do a typical bacterial plasmid extraction, after the bacterial culture was incubating overnight on a shaker plate. Afterwards, to assess purification success and to measure DNA concentration in solution, we inserted samples into a spectrophotometer and then constructed a standard curve. We then compared the values obtained from the bacterial sample to measure DNA concentration, and found we had a sufficient amount of DNA.
• Friday: In the morning, after verifying that the plasmid was correctly transformed, the next step is to reintroduce the mutated VHL vector into 786-O (a cell line isolated from a renal cell carcinoma) VHL knockout cells. Then, we will measure HIF-2A levels expressed in the cells after staggered time periods, to see the downstream effects of a specific VHL mutation on HIF-2A expression. I also continued with my personal goal today - I read a paper from the Cancer Research Journal (June 2006) about targeting and analysis of specific proteins in the HIF hypoxia pathway. Their findings generally corroborated our hypothesis - that a VHL loss-of-function mutation would result in a constitutively expressed HIF protein, which is understood to contribute to cancer formation (via angiogenesis, proliferation, and increased glucose metabolism). HIF is typically hydroxylated at specific prolyl residues and targeted for degradation by VHL ubiquitin E3 ligase, but in a pathway with either mutated HIF or VHL, this process can be disrupted. Additionally, hypoxia can inhibit this hydroxylation and result in HIF stabilization and subsequent translation of downstream genes involved in cancer development.

Week 3: (6/4/18-6/8/18)

• Monday: At first, I continued my reading today (when I arrived at lab, my mentor asked if I could wait a bit to start our experiment), with an article actually published by my PI (Dr. Zhuang). This article was especially interesting, because it had to do with specific patients here at the NIH, and their clinical presentation of >2 types of distinct neuroendocrine tumor, but with shared specific mutations in somatic DNA. After reading, I got started with my experiment for the day; I focused more on the HIF-mutated cells (E548K) mutation. We did a half-life/degradation study by adding MG132 (a potent drug that inhibits proteasomal degradation - effectively “freezes” the amount of protein contained in the cells), waiting 4 hours, washing with PBS, adding trypsin to break up the cells, collecting cell pellet, then freezing the cells at -80C for later use.
• Tuesday: We had a lab meeting today, where everyone shared their research - it was incredibly interesting to have the whole group together, and to share thoughts and ideas about each project. Additionally, most of the interns have arrived at this point, so I get to also learn about my peers’ research projects. Each Post-Doc shared their students’ research plans with the whole lab. Herui described my research, and talked about the known contribution of VHL mutations to development of cancer patients’ symptoms.After the lab meeting, I transfected the plasmid with the appropriate mutation into HeLa cell lines. The groups were the WT HIF, mutated HIF, and HIF A530V (a positive control). Each group was transfected with 1 ul red fluorescent protein (RFP) to test for efficiency after imaging. I used the established protocol for cell transfection with the Lipofectamine 2000 reagent and kit. After the waiting time was completed, I used trypan blue stain and our microscope, with a cell counter (Countess) machine to quantify the proportion of dead versus alive cells. Based on the concentration of living cells, I deduced that we could use 0.6 million cells per well (in a 6 well plate).
• Wednesday: The VHL experiment was postponed, since there was an error in the transformed bacterial vector. We sent a newly transformed bacterial vector for sequencing and hope (fingers crossed!) that it will come out correctly tomorrow morning! Just in case, later this afternoon, I will create another bacterial culture that has our transformed plasmid, to ensure we have something to work with. This included thawing the bacteria, adding the relevant plasmid, adding the mutant vector, letting the mixture incubate for 10 minutes on ice, heat shocking the bacteria to make them competent, adding SOC medium, then plating the bacteria on agar.
• Thursday: Today I retrieved the ctrl, WT, and E548K mutant cell lines after overnight incubation, and began my work with them. To help isolate the cells of interest, I spun down the lysate & agarose bead mixture, then removed lysate via centrifugation x1000g at ~10 Celsius for 3 min. I discarded the pellet and saved the supernatant, spinning the product in the cold room for 1 hr. After this process, I did the opposite - saved the pellet, discarded supernatant. I also began work on a protein degradation assay on this day, adding CHX, a toxic drug (protein synthesis inhibitor), to accurately measure HIF2A protein degradation over different periods of time - 15 minutes, 30 minutes, 1 hr, then 2 hrs. I also changed the medium for my HeLa cell line to work with later.
• Friday: Continuing my work with the protein degradation assay (half-life of HIF with and without mutations), I thawed my samples first. Then I sonicated the samples to break up the protein, boiled them at 100 C for 5 minutes, and spun them down. Then, I extracted my supernatant to keep, incubated again, let cool at RT, and then placed them on ice. Once this was done, Herui suggested that it would be most efficient if I ran a gel for analysis of both the CHX-treated cells for a half-life study and the results of a Co-IP assay for ubiquitination of a mutated protein. Herui walked me through the process of running and analyzing a gel, which was really useful!

Week 4: (6/11/18-6/15/18)

• Monday: We continued with the bacteria that I plated last week today. It needed to incubate a bit longer to show the mutation (which Herui proved by sending the mutant bacterial DNA for sequencing at the facility). I went through the protocol for bacterial plasmid extraction and analysis (working with the M54I VHL mutation), and brought the samples to the large centrifuge in Building 37 (which is directly connected to mine via a tunnel underground), and spun the samples at 13,000 rpm for 2 minutes. I then followed the Qiagen Quick-Start protocol for bacterial plasmid isolation, which included elution of the bacterial sample (after washing with a special buffer provided by the company) through a Qiagen tip and then elution of DNA with isopropanol & centrifugation.
• Tuesday: I transfected the HIF2A mutant E548K cells today for a second time. Herui explained the purpose of the 768-O cells and their treatment with puromycin. Only the puromycin resistant cells will survive, and the mutant vector was co-transfected with the puromycin resistance gene, so this will weed out only the cells with the mutant vector present. However, we added the lowest concentration of puromycin necessary to kill non-resistant cells. Once the cells were seeded to confluence, I added the OMEM medium and then added the mixture to the Lipofectamine reagent, then incubated. I then added the DNA-lipid complex formed to the cells, which will end in transfection of the target vector, and I will leave them for 1-several days until we can analyze.
• Wednesday: Today I analyzed the results of the transfection, and found that 95% of the cells were alive still (using the Countess machine with trypan blue staining to stain the dead/ruptured cells). I determined that with a well number of 5, I could use about 1.5 mL solution per group, which would result in about 500,000 cells per well. I passaged the mutant cells (786-O) that we transfected the HIF mutant into. The steps are shown:
  • Retrieve cells from incubation chamber
  • Pre-warm DMEM medium, retrieve trypsin + DPBS
  • Aspirate medium, wash w/ PBS, aspirate, add trypsin to break confluence on 6-well plate
  • Neutralize trypsin with DMEM, add solution to 15 mL tubes and centrifuge
  • Aspirate supernatant & resuspend w/ DMEM
  • Count cells

I also imaged the puromycin-treated cells to determine if they are still living, and the cells treated with 4 ug/mL had mostly died, but 2 ug/mL were usable.

• Thursday: I started the day by checking both of my cell lines (786-O cells and HeLa). Herui and I agreed that it was best to wait until Friday to do a Western Blot analysis for the degradation/half-life assay. I changed the medium (group by group), and added more mutant plasmid to each group to up the transfection efficiency. I created 4 groups:
  • 1. VHL WT w/ puromycin-resistant gene
  • 2. VHL Mutant
  • 3. RFP + puromycin resistant
  • 4. ctrl

I followed the same transfection protocol as before with the Lipofectamine (LFA) reagent. I am beginning to learn that research can take a lot - and I mean, a lot - of repetition!

• Friday: Today was not as busy of a day; I had several tasks to do, but many had long waiting periods. I sent the VHL mutant vector for sequencing to double-triple check that we have the right sequence. I spent some time today planning out the rest of my research plan and reading the relevant literature (much of which has included the input of current and previous members of my lab team). Today I completed the assay with CHX-treated cells to see the HIF half-life over time between mutated and non-mutated groups. This included the novel mutation as well as previously-studied mutations in order to have a positive control. We previously determined that a non-lethal dose of CHX for the cells in our assay would be 40 uL of CHX per well. The most busy part of the day was collecting the data from the initial, 15, 30, 1 hr, and 2 hrs intervals, as the first two intervals were relatively close together, and the data needed to be collected quickly as work was being done. The lab was relatively quiet today!

Week 5: (6/18/18-6/22/18)

• Monday: Unfortunately I was not able to make it to lab today, due to unforeseen travel circumstances; I was out of town over the weekend, and experienced a travel delay.
• Tuesday: Today I ran the Westerns for the HIF2a WT versus E548K mutant, with the Ubiquitin tagging assay, combined with the product of the CHX treatment (over several intervals) from Friday. It was a long and exhausting day, but I’m really hoping that at this point we will get some solid data out of this. Even though I have not been working with the VHL M54I novel mutation for a while, I’m excited to get back to that study and work on parsing out the characteristics of cells with that new mutation. I’m finally starting to really see how small experiments in the lab can have such a large impact, and can aggregate to form the basis for real clinical treatments and, eventually, patient outcomes. I set up the wells in the following way:
  • 1st gel (10 well):
    • 5 uL each WT sample
    • 2 uL ladder
    • 5 uL each E-K mutant
    • 4 uL ladder
  • 2nd gel (10 well):
    • 5 uL (WT)
    • 4 uL ladder
    • 5 uL 36 treated
    • 2 uL ladder

After planning the wells, I loaded the gel, put the running apparatus into an ice water bath to optimize the gel running conditions, and let it run for 40 minutes at 200V. Afterwards, I immediately ran the Western Blot and prepared the blocking solutions to save for later. Once this was done, it was the end of the day, so I placed the 6 well chamber with the Western Blot sheets on a shaker in the cold room and left it overnight.

• Wednesday: Today was a busy day! I started the day out by immediately coming in and continuing my Western Blot protocol. The steps are shown below:
  • Retrieve samples, preserve actin/HA antibody solutions (blocking buffer I made yesterday to incubate the WB strips in overnight)
  • Wash with TBST/shake for 5 mins/repeat 4x
  • Prepare secondary antibody solution for 2nd incubation
  • Add secondary antibody, shake for 1 hr at room temp
  • Repeat step 2
  • Image blot!

However - I ran into an unforeseen issue today, which was disheartening - for our WB analysis, we have two different substrates, the normal substrate, and the “supersignal” substrate. We used the stronger “supersignal” substrate, but still did not get a positive result (we could not visualize the bands, which are supposed to fluoresce bright blue). I asked Herui what we should do in this situation, and he suggested that I use a pre-made “stripping buffer” to essentially reset the WB incubation process, and then re-do the steps we did today on Thursday. We came to the conclusion that either the primary or secondary antibody was likely bad, and Herui claimed it was likely the secondary antibody. More tomorrow!

• Thursday: As hinted yesterday, I re-did the protocol for incubating the Western Blot starting first thing in the morning, but actually started with the primary antibody, let it incubate for 2 hours, then did the secondary antibody all in one day. This made for one busy day, and during the open periods I continued reading the literature, working towards my goal of reading some of the literature each week! Also, today I used the Anti-Rabbit instead of Anti-Mouse (which is the Antibody that failed yesterday). After this procedure, I tried to increase the transfection efficiency of the 786-O cells transfected with the M54I mutation in VHL. My overall aim is to see if HIF will be efficiently degraded by the mutant VHL protein (is the mutation loss of function?). We’re comparing the HIF levels of the WT and mutant cells with the 786-O cells that are VHL knockout, and will thus have higher background HIF levels, since VHL’s usual function is to tag HIF for the proteasome. Instead of using puromycin this time, I elected not to use it (since too many cells were killed).
• Friday: I continued the cell transfection that I left off with yesterday. Afterwards, I passaged the HeLa E548K cells, and followed the same steps that I followed the last time I did the passage. I started with pre-warming my DMEM medium & trypsin, then aspirated the medium from the cells after retrieving from the incubation chamber. Another thing I’ve also learned is that EVERYTHING must be sterilized in the cell culture room - the Postdocs are very adamant about cleaning gloves and equipment before and after leaving the room - which, of course, is important, so that none of the sensitive samples get contaminated. I washed the cells with trypsin to break them off of the bottom of the 6-well plate and then transferred them to tubes after mechanically shaking the plate (tapping it a couple of times) to loosen the cells and ensure they would all be transferred.

Week 6: (6/25/18-6/29/18)

• Monday: Today I began to try and replicate my results for the CHX assay with HIF degradation. I ran another Western Blot for this data, specifically using the samples from the 2 hour time interval (the longest interval I tested) because the HIF degradation showed the most clearly from this time interval. To re-test and amplify this result, I used the same cell line and process as the previous assay, in order to keep as many variables constant as I could. As a budding scientist, I am realizing that many times you need to make creative and innovative decisions to craft an experiment; it never truly is the “same thing day to day”. Herui and I worked together to analyze the result from earlier this month, and decided that the best result came from the 2 hr interval, and moved forward from there. This week was also interesting because a new girl joined our lab, who is in high school, but applied to our lab so she could gain some shadowing experience and learn more about the lab environment. It reminded me of when I first worked at NIH; I did a lot of shadowing (I was 16), but it was less laboratory shadowing than clinical shadowing - I assisted with patient records and attended clinical rounds, saw patients, shadowed surgeries, and more. I told the new student all about my experiences, and she was fascinated to hear about my journey from there - after Junior year of high school - to now. It was really fulfilling to have another person learning from my work and my experience; I hope she will go far and succeed!
• Tuesday: As usual, we had our lab meeting today (we always have lab meetings on Tuesdays; I haven’t written about it every week, but almost every week we do have one). These meetings are usually a lot of fun - for one of the meetings, the interns were able to share a short Powerpoint, put together by themselves and their respective mentor/Post-doc, and share their research project and beginnings of results with the rest of the lab. There are now about 6 interns in my lab (!) which is quite a bit for a lab with only 4 Post-docs. I really enjoy spending time with the other interns and hearing about how all of our research fits together with a special synergy and contributes to a greater goal. After our lab meeting, I was able to image the blot that I prepared yesterday, with the new student watching (name omitted for privacy purposes). Although she has not had previous lab experience, I tried to explain all of the procedures in detail to her, and how each one fit into the bigger picture. As a TA and previous tutor, I really love to teach and watch others learn, so this was a particularly special experience for me. I continued the M54I mutation experiment with a new luciferase assay, which measures gene expression at the level of transcription. Along with the cells expressing luciferase, I had 5 groups - a control group (no transfection), WT cells (two groups - one with MG132, which is a proteasome inhibitor to prevent HIF degradation, and one without), and then M54I cells (same two groups as above). I stored each group in a separate 10 cm dish in the cell culture room incubator and allowed the cells to sit overnight.
• Wednesday: After incubation overnight with (or without) the MG132, we checked the cells under the microscope to ensure they were still living and were confluent, then transfected another set of HeLa cells for study. I did two transfections today - one of the HeLa cells, and then the 786-O cells. There was some more repetition today, simply to try to replicate previous results. It looks like once we finish these assays, that we should have some reliable results to fall back on and use. The new student watched me again today, and I’m really enjoying having someone to talk to while I work - the waiting times do tend to get a bit long in research!
• Thursday: WOW - today was an incredibly fulfilling day! While I’ve been at NIH, I’ve been participating in the NOB-TRIP, which is the Neuro-Oncology Branch Translational Research Immersion Program. They offer several opportunities for the branch’s students to grow both as researchers and in other professional fields; each student is given the opportunity to shadow clinic at least once (which was my favorite part of working at NIH the first time!), my experience happened to have so much meaning to me. In fact, after I came back from shadowing and had some downtime during an experiment, I wrote an essay (that I can potentially use in the future for applications, or other uses) about a particularly touching experience I had meeting a family whose mother just learned her brain cancer came back - and that it could be removed, since they caught it early. Here’s an excerpt from my essay:

“That day, her family faced the last news that they would ever want to hear, yet held themselves, heads high, through heartbreak. I examined the two physicians closely, and took note of how their bedside manner shifted, ever so slightly. The slightest note of discomfort colored one’s face, in an internal skirmish between comforting her and maintaining clinical professionalism. The other gently patted her back.

I wondered how I would have handled a situation like this, had I been in either attending physician’s shoes. Self-restraint was essential. I specifically remember the word one physician – the chief of the branch – said to me as he described his intent: to “temper” the situation. He meant to provide a buoy to which a distressed patient could cling in an ocean of fear and doubt.”

I will undoubtedly take this experience with me into the rest of my career, and life. I finished up the day by checking my cells and changing the medium for the five 10-cm plates I prepared the other day. Our expected results for the luciferase assay (testing for HIF levels with different VHL conditions - mutated vs. not) are lowest HIF in the WT (no treatment or MG132), middle levels in the mutant, and highest in the positive control. I hope we can reach the results that we intend to - feeling hopeful.

• Friday: My two tasks for the day, continuing the experiments from earlier in the week, included passaging and freezing the extra (non-used) 786-O and HeLa cells for later use, after resuspending and adding 10% DMSO, then extracting HIF protein and examining WT/mutant expression of the protein. For the latter experiment, I had to create a RIPA lysis buffer and enhance it with PI/PMSF (1 mL of total solution). After aspirating the medium of the cells of interest, I added 2 mL PBS to each, aspirated again, then added 150 uL of the buffer to each group to break open the cells and measure the protein concentration. I brought the new student with me to the big machine in Building 37 (through the underground tunnel!) to sonicate the samples (she was shocked to find how LOUD this machine is - I let her use the earphones in the sonication room), then we came back to spin them down, then extracted and saved the supernatant. After this, I set up a BSA standard concentration with the spectrophotometer, and used a standard amount of my solution to measure HIF protein levels, compared against the BSA standard. During my free time, I continued working on my essay and spoke to Jared, a medical doctor who is new to the lab (working on a special project with my PI), and has incredibly interesting ideas about approaches to medicine, and is well-versed in developmental biology and how germline mutations affect later diseases. I really enjoy talking to him (we sit next to each other), and what he has to say about medicine/research always fascinates me.

Week 7: (7/2/18-7/6/18)

• Monday: Today marks the first day of my last week! Since Wednesday is a federal holiday (4th of July!), we have a day off. I’m excited to be able to wrap up my research and work on solidifying some good data. Herui insisted today that we repeat the luciferase assay (as per usual), and I’ll be continuing my WB analysis for HIF, VHL, & actin expression in the M54I mutated cells. Additionally, Herui and I discussed how we would finish the Co-IP experiment with HeLa transfected cells (E548K) for level of Ubiquitin tagging. I don’t believe I went into detail here with the aftermath of the Co-IP procedure previously, so I have summarized below:
  • Add 150 uL of prepared buffer solution to each cell-containing 1.5 mL tube (prepared and frozen previously) - groups: EK +MG132, EK -MG132, WT+, WT-, ctrl
  • Leave on ice (5 min), resuspend, ice again for 20 min
  • Sonicate samples in bldg. 37
  • Centrifuge at 4 C for 15 min; extract and save supernatant
  • BSA standard curve; compare protein concentration
  • Repeat WB protocol (as stated before)
    • Use HA Covance antibody for Ubiquitin
  • Finish WB Protocol tomorrow
• Tuesday: I did not finish the WB yesterday (since there are longer waiting times, Herui suggested I finish it today), so when I first got in I retrieved the samples, loaded the gel (with the Co-IP input, Co-IP product, as well as VHL M54I cells from before - we had a total of 16 samples, so I ran two gels; one 12-well and one 10-well, including the ladders). We had a lab meeting again this morning, so I let the gel run for 40 minutes at 200V in the cold room, and the lab meeting ran longer than this, so I retrieved the gel a little while after it was done. Technically the NIH is closed tomorrow, but Herui said he was going to come in (!) anyways, so he is going to help me out a bit to make sure I can get some results before my time here is done. Even though we’ve had to repeat several of the experiments, this is mostly to get replicated data, and I’m hopeful that I will have some solid results once the Ubiquitin study is finished. So, after I ran the gels and took the Western today, Herui is going to image the Western tomorrow, and we’ll discuss the results on Thursday when everyone is back in lab.
• Wednesday: No lab today - Independence Day!
• Thursday: Much of the same today, I spent some time discussing my project with Herui, and talking to him about how I could present my data. Although I am taking a summer course immediately after this internship and may not be able to make certain presentation dates for official NIH events, I am hopeful that I can present my research in other venues, potentially later on. This log is a great way of capturing the essence of my internship and giving myself a mode to remember all of the special things I have learned. Herui shared the WB results with me, which are a bit difficult to read (since Ubiquitin is so large!), but hopefully I will be able to show that Ubiquitin levels are decreased in mutated VHL, since the protein is typically responsible for tagging HIF for the proteasome. Again, Herui suggested I strip the WB and try another antibody, which may yield clearer results.
• Friday: My last day in lab! It’s been half of summer, yet it has gone by so quickly! Today I discussed with Herui some more details about presentation - the data, all together, could be described with the title: “Novel Mutations in Hypoxia Signaling Pathway Lead to Polycythemia” we discussed for a while what the best way to capture the research’s purpose would be - instead of giving primary attention to the mutations alone, we are giving a nod to the real patients who had these specific mutations, which guided the inception of this study in the first place. We worked on talking about some figures for our new data, and though they are not entirely finalized, they will be before the end of summer. I said goodbye to my fellow interns today and wrapped up my last experiment!