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.

After learning what we needed to know about creating polyacrylamide gels, our task was simply to create simple procedures for, run, separate, and analyze a number of reactions testing the reactivities of a few commonly used proteins in our lab; the reducing power of our reducing agents; the effectiveness of the materials we use to halt our reactions; and the labeling power of our dansyl chloride-labeled glutathione under different conditions of concentration, time, and acidity (pH). We have been using two proteins for these reactions, lactate dehydrogenase (LDH) and creatine kinase (CK); these proteins seem to respond well to our fluorescent labeling reactions and give us the clearest results when they are analyzed on the gels as, when labeled, they produce bands that are tight and highly-defined. (We tried using one other protein, papain, but found it difficult to analyze because it appeared as a large, dark smear on the gel.) Through our reactions we have found that, as we predicted, more reduced, unlabeled protein converts to oxidized, labeled protein with increased exposure to (increased concentration or time with) DGSSG and DGSSGD. We are extremely happy that things have been working out this way. So far, dansyl chloride-labeled glutathione products have been giving us better results than fluorescein-labeled glutathione products ever did.

As part of these experiments, we also have to analyze the differences in labeled protein concentrations that arise from protein reactions with DGSSG and DGSSGD. We have primarily been working with DGSSGD during these experiments, as it produces the most labeled protein on the gels; but we have also found that DGSSG does seem to label a good bit of protein itself. (Exact concentration comparisons are unavailable to us at this time because, as I stated in my previous blog post, we are unable to convert fluorescence readings into concentration values because of the quenching patterns of the fluorescent labels on the molecules. We have been working off of mean fluorescence intensity value readings for our comparisons of the amount of labeled protein we see.) Fortunately for us, though, our analyses so far have caused us to believe that proteins do not seem to favor one labeled end of the DGSSG molecule over another in its reactions, a steric problem that was readily apparent through prior reactions with fluorescein-labeled glutathione (FGSSG and FGSSGF). (We have been constantly comparing the reactivities of DGSSGD and FGSSGF with our proteins to reconfirm this conclusion.) And so far, the labeling patterns we have discovered with reactions between DGSSGD and CK or LDH hold true for those same reactions with DGSSG. The only problem we have encountered is that of identifying a large dark smudge that appears below the labeled protein band when DGSSG is reacted with protein and run on a gel. We have yet to identify it, but for the purposes of our reactions so far it has not been a huge issue—the same smudge appears when DGSSG is run on the gel alone.

As we began our time trial comparison reactions, we started using iodoacetic acid (IAA) to bind to extra thiol groups and stop our reactions in their tracks. However, when we ran control samples with IAA added before the DGSSG or DGSSGD had been added to the reactions (in an attempt to prevent them altogether), we found that the reactions had still proceeded to a small extent, even when higher volumes of IAA had been used and even though the concentration of the IAA should have been more than sufficient to halt the reactions. As a result of this, we tried using iodoacetamide (IAM), a similar compound, to see if it performed any better than IAA in this capacity. We set up reactions with LDH and CK in combination with DGSSGD reactions, adding IAA and IAM before adding the DGSSGD and letting the reactions sit for different lengths of time in order to prevent the reaction from occurring; we then allowed the reactions to “run” for ten minutes before moving the samples to the gel. Overall, IAM is more effective at both preventing our protein reactions and at stopping them after specified periods of time. It appears to be most effective within about ten or fifteen minutes, beyond which time letting the reaction sit with IAM does little to help prevent or stop it from taking place.

Once we conducted the reactions mentioned above, we had a general idea of what each of the basic reactions would look like on a gel and what materials we would need to conduct similar reactions under a variety of other conditions. This allowed us to move on and study some more complex reactions, which I have described below:

– Fifteen microliter reactions: Most of the reactions that we have run in the gels have required about ten microliters’ worth of reagents. In these reactions, we decided to try using fifteen microliters’ worth of reagent material in order to produce clearer results on our gels.

– Reactions with DTT: In order to ensure that the bands we assumed were labeled protein were indeed labeled protein, we attempted to re-reduce the protein and remove their fluorescent labels to make the bands disappear. By conducting two types of reactions with DTT, one in which we varied the concentration of DTT used and one in which we varied the amount of time for which we ran the reactions with DTT, we discovered the following patterns: with increased amounts of DTT used we saw less of the labeled protein; however, additional reaction time with DTT made very little difference in the amount of labeled protein observed. These types of reactions were conducted with both DGSSGD and FGSSGF.

– Acidity tests: As a crude version of the reactions we intend to conduct a little later on, we decided to compare reactions between DGSSGD and LDH and between DGSSGD and CK at two different pH values—one at a pH value of 7.4, and the other at a slightly higher pH value due to the addition of phosphate buffer pH 8.0 (in place of phosphate buffer pH 7.4) and DGSSGD at pH 8.0 (in place of DGSSGD at pH 8.0). We found that the change in acidity made little difference in the reactivities of the proteins and DGSSGD.

– Percentage DGSSGD and FGSSGF concentration reactions with GSSG: In order to ascertain whether or not the proteins selectively interacted with GSSG based on the presence of a fluorescent label, we decided to mix different ratios of DGSSGD or FGSSGF with GSSG and allow the reactions to proceed as normal—100% DGSSGD or FGSSGF, 80%, and so on, until we reached 0%. We were to infer that no preferential reactions took place if, as the concentrations of DGSSGD or FGSSGF decreased linearly, the amount of fluorescently labeled protein on the gel would also decrease linearly. This is indeed what we found with DGSSGD, and to a lesser extent with FGSSGF.

– Protein concentration variation: In our early reactions with the gels, our lab decided that, in order to only have one variable influencing our results at a time, we would adjust the concentrations of DGSSG, DGSSGD, FGSSGF, and GSSG in our reaction mixtures. We wanted to see what would happen should we alter the concentrations of our proteins instead. Would the proteins compete and react with one another at higher concentrations, diminishing the overall amount of labeled protein product? When we actually conducted these reactions, we found that this was not the case. The amount of labeled protein product simply increased linearly with higher concentrations of reactant protein.

After all of this, we conducted a few more reactions to spot on thin layer chromatography plates. These included reacting GSH with labeled DGSSG, DGSSGD, and FGSSGF as we did before to see how the amount of labeled glutathione that appeared on the plate would change. We also reacted DGSH with hydrogen peroxide, an oxidant, to try to eliminate the spot of labeled DGSH that typically appears when it is run on a TLC plate. After conducting many of these reactions with different reaction times and concentrations of hydrogen peroxide, we found that about fifteen microliters of the oxidant and twenty minutes of reaction time were required for the DGSH to all but fully re-oxidize and eliminate the DGSH spot on the TLC plate. We then decided to add DTT, our primary reducing agent, into the DGSH-hydrogen peroxide mix to see if we could re-reduce it and make the spot reappear on the TLC plate; we were excited to find that we could! We could essentially switch the spots back and forth on the plate at will, though we do not yet know whether or not doing so caused any damage to the glutathione molecule itself. We will continue to study this later.

Overall, it has been an exciting three weeks. We have done a lot of work, but there is still much left to do. I plan on staying on campus for an extra week now, thereby committing myself to a full eight weeks of research rather than seven. We will see what happens!

Comments

  1. Wow Taylor, that sounds awesome! Obviously I don’t understand everything that you are doing even though I am doing chemistry stuff as well, it seems like you have made a step in determining what methods works best and where certain bands in the gel show up, which is a big step in being able to continue with research. Although I am doing something completely different, I realized that we use the same types of techniques, like TLC, to determine what reactions look like and how we can spot them in other conditions. I have found this technique incredibly helpful since I have been running columns and have not been able to find my product, so I am able to relate to the sometimes frustrating and confusing process. I also think that it is awesome that you discovered something that you can look into in the future using TLC plates — makes me think about things that I could do!