Working with Dansyl Chloride-Labeled Glutathione, Blog Post #3: Wrapping It Up

Hey, everyone! It’s been a good while since I wrote my second blog post, and it’s actually been a couple of weeks since I finished working in Dr. Landino’s lab for the summer. During the interim I’ve been gathering and organizing data from our experiments up to the eighth week of the summer (a period extending from May 31 to July 20): this includes creating data tables detailing our experimental concentrations and fluorescence intensity results as well as organizing our photographed images and notes into a long slideshow. At present I’m working on creating a final, large data table that displays the general results and reactivity trends we observed over the summer; the table will make developing a display for the Freshman Monroe Grant project exhibition a much simpler task.

While at this point I cannot give much away as our results, I’ll explain what we did during my final two weeks on campus. Towards the beginning of that time, we performed a set of experiments with mixed DGSSGD (D2) and GSSG samples similar to what we had done previously, where we varied the ratios of D2 and GSSG concentrations and observed the resulting changes in protein labeling on the gels. On top of that, in order to begin tackling the major problem we’ve faced of relating fluorescence intensity values to concentration values, we placed samples of known concentrations into the wells of 96-well plates, photographed them, and measured with a computer program the fluorescence intensity values of the compounds in the images (just as we had been doing with our gels). These sorts of measurements were performed with five different dansyl chloride labeled compounds and one fluorescein labeled compound. Comparisons were then made between the compounds, and we constructed plots demonstrating the relationships between compound concentration and fluorescence intensity. We may be able to use these plots to help us estimate the concentration values of our labeled proteins and glutathione on future gels and plates.

Following these experiments, we decided that we would continue our studies of DGSH’s oxidation and reduction cycles by performing such cycles while varying reaction time. We also set up reactions in which we combined DGSH or FGSH, creatine kinase (CK) or lactate dehydrogenase (LDH), and an oxidant in order to encourage the reduced labeled glutathione molecules and reduced proteins to react and create labeled protein bands on our gels. We performed many sets of these reactions in which we varied reactant concentrations (namely of the oxidant or DGSH or FGSH), reaction times, and/or the orders in which the reactants were added to the reactions (as we generally allowed the first two reactants added to react for ten minutes before adding our final reactant). We also began testing and comparing the dependence of certain reactions on the acidity of the surrounding solutions by adding phosphate buffers of various pH values. Unfortunately, the results we gathered from this last set of experimental reactions were fairly inconclusive.

Our final task at the end of the eight weeks, aside from organizing the data we had collected, was to perform a set of reactions that could be used to summarize the results of the experiments we had performed. Our lab lovingly described the photographed gel images we produced as “The Story of Creatine Kinase and Dansyl Chloride,” as they, while not nearly marking the end of our work, served to demonstrate just how much we had accomplished over the summer, despite the fact that we were unable to reach a point where we could feasibly work with tubulin.

Overall, it turned out to be quite the busy time, and I wouldn’t have traded it for the world. I benefited greatly from the ample amount of valuable laboratory experience and was able to really bond with the people working in my lab. Before I end this third and final post, I want to thank Dr. Landino for providing me with the amazing opportunity to work in her lab and support her research, and Dr. Landino, Sharon, and Olivia for making the summer so informative, light-hearted, enjoyable and rewarding. I look forward to continuing the Landino lab’s studies during the fall semester!