Reflections

I’m writing this blog post as I sit in lab during my last hour incubation period of my last CAT ELISA on the second to last day of the summer research term. This summer has been a roller coaster ride. I’ve experienced incredible highs thinking that an experiment would finally work with one small tweak to the procedure and dramatic lows upon spending nine hours on a small sample size and a tiny plate only to find that the ELISA was unsuccessful. At the very least, research this summer has allowed me to become quite familiar with the CAT ELISA procedure and I think all of the small errors and kinks, from inverting trypsin tubes when seeding plates to partially incubating plates at room temperature with lysis buffer to improve protein yields, have been worked out. If I continue CAT ELISAs in the Fall, I don’t think I’ll have to spend a lot of time trying to fix procedural problems. I consider working through the procedural problems alone to be a success regardless of how much, if any, data is collected from a modified procedure; however, it would be really nice to actually collect some data from a proper experiment at the end of this research term. Unfortunately, there isn’t enough time to note whether or not results from these ELISAs form a trend so hopefully a pattern will start to emerge in the Fall after more modified CAT ELISAs. On the upside, besides carrying a specialized knowledge of how to perform CAT ELISAs away from this term, I’ll be able to apply more universal techniques I’ve learned this summer to future lab work. I feel more confident in my flask-splitting and plate-seeding technique and also more comfortable with transfections. Now onto another success-defying CAT, the MCATs…

It’s all about Media, Media, Media

One has to admire the sheer amount of media required for tissue culture work. In the process of splitting cells, eight milliliters of fresh media is added to the old flask and only a small amount of this media, containing the cells that had reached a high confluency in the old flask, is added to a new flask containing around 15-20 milliliters of fresh media. Each plate in a CAT ELISA requires one milliliter of the resuspended cell mixture and nine milliliters of fresh media, if using a flask with proper confluency for seeding plates. The remainder of the resuspended cells in the fresh media and old flask are aspirated off, never to be used in a transfection or CAT ELISA. Each media change requires ten milliliters of fresh media to be added to each plate, so media changes alone require 120 milliliters of fresh media over the course of less than 24 hours when using six plates. I recently incubated some of the media I have been using for CAT ELISA transfections and found that nothing grew in it, suggesting that it’s not contaminated. This lack of contamination leaves the question of what is causing so many cells to detach from the plate surface post-transfection. It seems as though 70-80% of the cells present on the plate pre-transfection are being lost after tranfection and media changes. Transfection is a harsh process, however it seems unlikely that only 20-30% of the cells could survive. During my last transection, pre-warming the media seemed to help maintain higher confluency, but the vast majority of the cells did not survive the process. A low numberof surviving cells contributes to the low protein concentration issue so eliminating or mitigating the influence of this source of rampant cell death and detachment would be immensely helpful in the pursuit of elusive reasonable results. Considering the lack of time left in this summer research term, the next time I split a flask of cells, I plan to use the entire resuspension amount to seed two sets of plates and perform identical transfections for both. If the protein concentration from both transfection sets is high enough and the ELISA results are reasonable, I’ll be a little closer to CAT ELISAs with thyroid hormone receptor beta.

Odds, Ends, Protein Concentrations, and CAT Standards

CAT KILLER STRIKES AGAIN! My latest victim bore the additional burden of the lowest protein concentration of any transfected samples thus far. Could this be due to the room temperature lysis buffer incubation I had performed? I’m not sure. I would be able to narrow down possible causes of this low protein concentration had I done what was suggested (take a 10 microliter portion of one of the samples after the thirty-minute lysis buffer incubation and observe whether or not the cells looked whole). I forgot this step in a rush to determine protein yield with high hopes that this would be my first successful CAT ELISA with a high protein yield due to the room temperature lysis conditions. Since CAT ELISAs depend on cell lysis for effective measurement of protein concentrations, whole cells appearing after the incubation with lysis buffer would lower measured protein concentrations. Subsequent centrifugation of whole cell samples would cause high levels of protein loss as the protein would pellet out contained in the cell. Instead of wasting plasmids on a process that my or may not work, the next step is seeding normal HeLa cells in plates and subjecting different plates to room temperature, ice and half-room temperature/half-ice lysis to determine which conditions are least conducive to whole cell state maintenance. Another recent critical change to the protocol is the use of 10 microliters of thyroid hormone rather than the one microliter amounts previously used; if I ever proceed beyond the Bradford Assay again, this larger amount of thyroid hormone will hopefully solve the issue of no apparent thyroid hormone induction. Unfortunately, I’m also still getting exponential CAT Standard curves where they should be linear. I’m not sure how to fix this problem besides trying to epitomize vigorous pipetting to ensure uniform CAT Enzyme distribution.

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Troubleshooting

Start of the third week and back to performing CAT ELISAs. I am constantly reminded of how important getting the details right is to successful experimentation. Some of these details that I have failed to remember or perfect in past and current CAT ELISAs are: making sure the protease inhibitor tablets have completely dissolved in the lysis buffer before adding the lysis buffer to the plates, inverting the CAT Enzyme Working Dilution tube multiple times and pipetting the solution up and down to try to ensure a uniform distribution of CAT Enzyme within throughout the solution, making sure the cold centrifuge is set at 4˚ Celsius, checking the multi-channel pipette to ensure that each pipette tip has actually taken up solution, loading the sample trays in the CAT ELISA plate in the correct orientation so the plate reader will read them, and famously (from my first attempt at a CAT ELISA), not using working dilutions as washing buffers. These are only small parts of the CAT ELISA procedure, but remembering and performing them makes all the difference in the world.

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Waiting for my cells to divide…

Re-entry into lab work has proven to be an interesting challenge. I arrived on campus this past Sunday and have been analysing data from CAT ELISAs performed during the semester and performing plasmid preps to stockpile plasmids for use this summer. This break between the end of the school year and the beginning of the Summer Research Term is the longest I’ve had since I entered college; my catharsis regarding breaks is that it’s amazing how little time it takes to fall out of good study and work habits. I am still in the process of regaining or attempting to regain the same level of focus I had during the semester and have found that the transition to a more studious approach does not occur with the same ease that flicking a switch does, however, it’s a relief to be able to come back and devote time to research, rather than splitting time between research and classes which always happens during the semester. The concentration that studying and working requires also brings a sense of quietude and serenity that is unparalleled in any other tasks. The first week back to research passed quickly and was consumed mostly with administrative tasks and planning aside from data processing and plasmid prepping. Plasmid prep brought up some pertinent questions for future transfections. When the prep yields high concentration samples, should they be diluted down, requiring a higher plasmid transfection volume and increasing the likelihood that the plasmid will disperse throughout the transfected plates, or should the concentration be kept at a high level and lower volume? Plasmid dilution may introduce impurities while smaller volumes of higher concentration plasmid may not disperse throughout a transfected plate as well as the higher volume, lower concentration plasmids. I will be performing a CAT ELISA this week to determine if the data from previous ELISAs was valid and this will hopefully give me a point of comparison to determine whether or not plasmid dilution is worth the risk of impurity introduction or if this step in the process even effects a significant difference in the ELISA results. If the variability in results from the past ELISAs can be lowered over the course of the summer, I plan to move onto CAT ELISAs with Thyroid Hormone Receptor Beta (I’m currently only working with Thyroid Hormone Receptor Alpha to minimize the chance of error introduction which can occur when working with larger samples). The purpose of the CAT ELISA is to quantify the amount of CAT Enzyme expressed in transfected cells. Experimental cells are transfected with both a Thyroid Hormone Response Element (TRE) and the suspected exportin; the TRE is necessary as it is the site of Thyroid Hormone Receptor binding. Thyroid Hormone Receptor can bind to the TRE regardless of the presence of Thyroid Hormone (T3), however T3 presence generally allows the receptor-hormone complex to act as a transcriptional activator. As a transcriptional activator, transfection sets are performed as duplicates with T3 added to one of the duplicates, allowing comparison with T3 deficient samples. If the protein acts as an exportin, data would show decreased levels of CAT Enzyme expression relative to cells that were not transfected with the protein. Analysis of these transfected cell lysates after performing the CAT ELISA protocol and comparison with control cell lysates will enable characterization of the protein as an exportin or demonstrate its deficiency in this area of function. The long break between the end of last semester and the beginning of summer research has also meant needing to cultivate more cells to work with and unfortunately, cell growth doesn’t just occur when one dangles colchicine over a flask and threatens the cells to divide, it involves waiting for the cells to divide and reach the proper confluency. Until then, I’ll be updating my lab notebook, reviewing literature and protocols, and getting to know every cracked spine and bent page in my MCAT books (and hopefully next week I can forget all of these features while transfecting and performing ELISAs).