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…

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.

[Read more…]

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).

Nucleocytoplasmic Shuttling of Thyroid Hormone Receptor

Hello, my name is Mary Stern. I am a rising senior and Biology Major. Over the summer I will be working in Dr. Allison’s Lab to try to clarify the role that exportin 7 may play in Thyroid Hormone Receptor Export from the Nucleus. Thyroid Hormone Receptor can shuttle between the Nucleus and Cytoplasm of a cell, enabling it to impact gene expression through actions as a transcription factor. Perhaps, a step should be taken back at this point to explain some of the information above in a little more depth.

[Read more…]