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.

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Working with Dansyl Chloride-Labeled Glutathione, Blog Post #2: Introducing the Polyacrylamide Gel

Hey, everyone! It’s been quite a while since my last post, so it’s high time for me to give you an update as to what has been going on in the Landino lab. Well, once we finished trying to re-oxidize reduced dansyl chloride-labeled glutathione (DGSH) and reduce oxidized single dansyl-labeled glutathione (DGSSG) and double dansyl-labeled glutathione (DGSSGD), we began working with polyacrylamide gels. These gels are especially important in our lab, as through them we can separate the proteins and materials we regularly use in our reactions. Once separated, any fluorescent labels or labeled material (often protein) in the gel can be visualized (usually as bands or smears) with a specialized camera-like apparatus we have in the lab. While extremely useful, the gels take some time to make, and it is always worrisome when we have to deal with acrylamide. (Acrylamide itself is a neurotoxin; polyacrylamide, fortunately, is not, so the hardened/polymerized gels are essentially harmless.) However, by now we have made and run reactions in these gels so many times that it is like second nature, and I frankly enjoy doing it.

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Working with Dansyl Chloride-Labeled Glutathione, Blog Post #1:The Preliminary Production and Testing of Dansyl-Chloride Labeled Glutathione

When Sharon, Olivia, and I walked in for our first week in the laboratory, we were not sure as to what our summer lab sessions would entail. Dr. Landino helped us prepare for the coming weeks, explaining our goals for the summer and clarifying our basic plan of action—all in all, we hope to run protein reactions and gels with the majority of our commonly-used lab proteins, comparing their reactivities under different pH, time, concentration, and temperature conditions. To do this, we would need to purify a lot of our primary protein target: oxidized dansyl-chloride labeled glutathione. So for the first couple of days, we spent our time purifying such samples using C8 columns. We loaded solutions of reacted dansyl chloride and glutathione onto our columns and followed with fractional washes of methanol, deionized water, and acetic acid. (Excess dansyl chloride elutes in 20% methanol, single-labeled oxidized glutathione elutes in 50% methanol, and double-labeled oxidized glutathione elutes in 80% methanol.) Though not exceptionally difficult, the column procedures were time-consuming; but we prepared a good amount of product using the column technique we finally perfected after a semester of trying to get it to work properly. Huzzah!

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Abstract: Determination of protein cysteine reactivity with fluorescently-labeled glutathione derivatives

This summer I will conduct research with Dr. Lisa Landino’s laboratory team into the potential damage of electron removal from molecules composing a key protein in cellular microtubules by reactive molecular radicals. This electron removal, also called “oxidation,” causes the protein’s subunits to unnaturally bind together, impeding the normal function of microtubules in cells. Since microtubules provide structural support in all cells and are especially important in nerve cells, their impairment may contribute to the development of neurodegenerative diseases such as Alzheimer’s and Parkinson’s. With this in mind, our lab team attempts to study the mechanisms behind neurodegeneration by radical molecules and determine whether or not this protein is involved in hopes of ultimately developing a means of reversing nerve cell damage.

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