The End is Quickly Approaching- Week 5

Just a reminder, my name is Colton and I’m visiting the Buccella Laboratory at New York University. The lab is part of the chemistry department. More information about the lab can be found @ http://www.nyu.edu/fas/dept/chemistry/buccellagroup/ .

As I mentioned in last week’s post, we were still testing the magnesium indicator in the presence of ATP. This week we attempted to determine whether the compound was photo bleaching and/or taking a while to equilibrate. Monday we started two experiments to attempt to answer this question.

To test the amount of time to equilibrate we took fluorescent spectra every 10 minutes, allowing the solution to stir and settle between spectra. This allowed for any complexes to form between the sensor, magnesium, and ATP. The fluorescence decreased as the solution equilibrated, which we thought may occur since we believe that ATP was quenching the fluorescence. However, we let another vial of the same solution sit all day and equilibrate by it, and when testing it at the end of the day, it had the same fluorescence level of the first spectra taken. This indicated to us that the solution freshly prepared does not take long to equilibrate, since it has the same fluorescence as a solution that was able to equilibrate all day. Thus, the steady decrease we saw every time would mean that the compound was photo bleaching.

We decided to do a time spectra for to test for photo bleaching. Every 2 minutes the fluorimeter automatically took spectra of the compound in buffer. This frequent exposure to light will break the compound down if it photo bleaches and show a slight decrease in fluorescence each time spectra is taken. Our assumption that the solution was photo bleaching seemed to be true, until approximately the 19^th scan where fluorescence began to increase with each additional spectrum taken! We were baffled by these results.

More or less, we were still left stranded trying to come to some sort of conclusion as to what the system was doing. Our only option left was to do an NMR titration to know what complexes were forming in the solution and hopefully answer why the spectra was going down then up.

To make sure we didn’t get incorrect data we repeated the experiments. We will not be able to start the NMR titrations until next week.

We went in this week with a lot of questions, and even though we didn’t answer any, our data will have meaning. After we figure out what complexes are forming we will hopefully be able to understand this week’s data and previous week’s data. I’m not looking forward to the start of next week, since it will be my last 5 days in the lab. I have learned so much about fluorescence and science in general. It won’t just be the end of a summer internship but also the end of many days spent with great people and many laughs all within the realms of chemistry. 

Fluorescence; The Crazy World of Wavelengths Week 4

Just a reminder, my name is Colton and I’m visiting the Buccella Laboratory at New York University. The lab is part of the chemistry department. More information about the lab can be found at  http://www.nyu.edu/fas/dept/chemistry/buccellagroup/ .

Monday morning started off early as usual. As I waited for everyone to arrive, I ate my breakfast overlooking Washington Square Park. It is such a beautiful view, but It isn’t just the view that is astounding; it’s a combination of NYU, their facilities and more importantly the friendly, intelligent people I’ve been able to work with.

Finally when Sarina arrived,(the graduate student I’ve been working with), I found out that I would be continuing my mini-project. My mini-project is fluorescent tests on 3 different compounds in various solvents, that I mentioned in “Fluorescence; The Crazy World of Wavelengths Week 3”. I had to finish testing compound 1c, which took up the morning and went into the afternoon.

The next day I planned to continue the experiment, but there was a catch. I've come to found that this often happens in research and it is generally time related. I could technically start, but I couldn't collect data till the following day. The issue was a matter of equilibration. Compounds 1a and 1b that I would now be testing had to equilibrate for at least 6 hours in 2 of the solvents I was testing them in, PIPES buffer and water. I prepared these solutions and all the others, so I would not have to take the time to make them the following day. After a quick lunch we continued our ATP titrations. A brief reminder; we are testing a fluorescent compound’s fluorescence when bound to Magnesium and the effects ATP has on the system. *For a more detailed background of the experiment please see week 2 and 3 of my blogs.  10 fluorescent scans had to be taken every ½ hour which meant we were going to be in the lab a while. Combined with our late start in the afternoon and the amount of time needed between scans, we didn’t finish the experiment until 8:30pm. It was all worth it in the end, we gathered the data we needed and found out that we don’t have to wait as long between scans in future experiments. On the overhand, we were still unsure what exactly was happening within the system. We believe it could be photo bleaching or slow equilibration. Photo bleaching is the breaking down of a compound over time as it is subjected to light (excited). Photo bleaching occurs at different rates for every compound, and the goal is for a compound to slowly photo bleach, meaning it can be excited multiple times before truly breaking down.  

The next day I was able to continue my mini-experiment on the UV-Vis & fluorimeter immediately. I have figured out a fairly proficient system for running my tests, which allowed me to finish them in a few hours.

Uv-Vis

Fluorimeter 

At group meeting this week, Debbie presented techniques used in the paper “Labeling Lysine Acetyltransferase Substrates with Engineered Enzymesand Functionalized Cofactor Surrogates”. You can access the article by clicking on its' title above. Although there was some information I did not have enough biology background to understand, I found that Debbie did well presenting the information, only adding to my comprehension. The big picture for me was learning more about the ways in which biology and chemistry interact.

Next week should be just as exciting as we begin to explore the reasons behind the results of the ATP titrations!

Fluorescence; The Crazy World of Wavelengths Week 3

My name is Colton and I’m visiting the Buccella Laboratory at New York University. The lab is part of the chemistry department and we are studying fluorescent compounds that bind with magnesium.

 This week went by pretty fast and I’m already sad that I only have 3 weeks left. Like usual I was up at 4:30am to catch the train and be in the lab by 9:30am. As soon as Sarina came in we began that day’s experiment; testing changes in spectroscopic properties of a compound in different solvents. You may be looking at your screen asking yourself “Well what does all that mean?”. Spectroscopy is the study of how molecules interact with radiation (light). We are testing the fluorescent properties of a molecule. Essentially how much and what type of light a molecule emits and how that changes according to the solvent it is in.  

First, we had to make our solutions using the compound 1C.

Compound 1C
(SCS_2_185 & CK_1_06)

Using DMSO (Dimethyl Sulfoxide) we diluted 10uL(micro-liter) of 1mM (milli-molar) compound 1C to a 2uM (micro-molar) solution. We did the same but in ACN (Acetonitrile). We then began our tests on the UV-Vis and fluorometer. Using the UV-Vis we were able to gather the absorption maximum of each solution. Absorption maximum is the wavelength with the greatest absorbance for a compound. That maximum is then used as the excitation wavelength to gather emission data on the fluorimeter. Basically, that wavelength of light is used to excite the molecule. The emission scan tells us the wavelength that is emitted with the greatest intensity when excited with that given wavelength. The emission maximum (the wavelength emitted with the greatest intensity for a given excitation wavelength), is used in the excitation scan. The excitation scan tells us what wavelength is necessary to have a compound emit a certain wavelength (essentially confirming the absorbance scan). It was unbelievable to see how the fluorescence intensity changed so much in different solvents. Not only did it decrease but it either shifted left or right on the light spectrum. Left shifts move towards blue light, and right shifts move towards red light.  Here’s a graph to give you a visual of the data we collect.

Absorbance Data of Compound 1C in Various Solvents. Peaks represent absrobance maximum.
(CK_1_06/16)

Fluorescence Data of Compound 1C in Various Solvents. 
(CK_1_06/16)

Monday was a long day and we didn't finish up until about 7pm. We were tasked that night to plan for an unplanned experiment to start the next day. So for the time being my experiment was put on hold, because we needed the instruments for this new experiment. The previous week I mentioned that Brismar and I were testing a compound, and as it turned out, we needed to check the compounds binding ability with magnesium in the presence of ATP. The way the compound was designed it only need to bind at two places with magnesium and magnesium has four binding sites. This may seem insignificant, but it’s not because this means magnesium can still bind with ATP, which is not beneficial to our research. The goal of the fluorescent sensors the grad students and Dr. Buccella are designing are to locate free magnesium in the cell, not magnesium bound to other compounds and proteins. So for the rest of the week, that would be the project that Sarina, Brismar and I would be working on.

Before testing anything, we had to make solution of ATP and Magnesium-ATP. It was much more challenging than we were expecting. The original concentration of ATP we wanted in each solution would require more ATP than we had. When we figured out the most concentrated solution we could make with the ATP we had, we didn't account for the Kd (dissociation constant) of ATP, which would not allow us to make the concentration we wanted to, because it wouldn't dissolve in such a little amount of solvent. After an hour or two of thinking, plus doing various calculations we found a concentration that worked. So Tuesday was spent making our solutions. We were finally able to begin the experiment. The first test we did was with the Magnesium-ATP solution. As we preceded with the emission scans, we began to see a decrease in fluorescent intensity as we added more Magnesium-ATP to a solution of the sensor. This was really odd. We figured the ATP binding with magnesium may quench the fluorescence a bit, but not decrease as we add more. Well we did some thinking with Dr. Buccella and we discovered that the equilibrium of the reaction was slow. So when we proceeded with the second trial, we allowed the reaction to reach equilibrium after each addition of Magnesium-ATP. It turns out this was all we needed to do, and instead of decreasing, fluorescence increased, but not as much as when ATP is not present, which means ATP is affecting the fluorescence. (Always do experiments twice… you never know what could happen.)  The rest of the week consisted of us repeating this experiment and doing the same experiment with just ATP, to see how the sensor is affected by the ATP, with without the presence of Magnesium.

To sum the week up, it was very exciting with many surprises. For me one of the best parts was seeing and helping figure out why we were getting the results we got and how we could test them further. It was a lot of critical thinking, but that’s never a bad thing!

    

Buffer, Learning and More Buffer- Week 2

I was awoken at 5 am by my alarm and by 5:30 my journey back to NYC began. I arrived to the lab a bit early and waited for Brismar to arrive so we could start analyzing data from the previous week. I found out we had to gather some more data since analytical chemistry requires multiple trials of the same experiment. This way the compiled data is the most accurate.

We thought we were finished making the buffer last week, but as it turns, in making another batch, that we may have added to much KOH. So to test our assumption we checked the pH and found that the buffer we made last week had a pH of 7.09 not 7. Even though it may seem like this is not a big difference, the pH that the solution is buffered at is really important. Especially when using it for our experiments, where a small change in pH could completely upset the fluorescence of a compound or its binding ability. In the ended we ended up having to spend two days making new buffer, but this time we carefully added KOH so that the pH was exactly 7.

This week NYU had a guest speaker in the chemistry department discussing bio-imaging, similar to what our lab does. With our professors suggestion, we all went to the seminar. I found the presentation extremely interesting. Him and his group are developing fluorescent probes that are able to detect different types of cells in the human body. The fluorescent sensors choose certain cells based on unique proteins and other characteristics unique to a type of cell. For example, his one probe detects stem cells, so when injected into an organism only stem cells would fluoresce. This able to show where concentrations of a type of cell exist. The goal is to be able this with all different types of cells.

In addition to this presentation, our professor gave us a presentation on presenting science, whether it be presenting research within our group or at a conference. Some of the major points I took from the presentation were 1) thinking about who your audience is, 2) what do you want them to know, and 3) make sure it's professional, not only so your audience takes you seriously but also takes your science seriously. She made it clear, multiple times, if you don't seem serious, then why is your science serious, or if you appear sloppy and your presentation is sloppy, then who knows if your science is sloppy.

The week ended with planning and preparing for an experiment Sarina and I will start Monday. We will be studying the fluorescence of three compounds in different solvents. I'm so excited to start another week because the learning never seems to end!  

Rehabilitating My Relationship With Chemistry -Week 1

After completing AP Chem this school year, Chemistry was one of the last things I wanted to talk about. However, after a couple hours of being in the Buccella Laboratory at NYU, I began to fall in love with the subject again.

I made my way to the 8th floor of the Brown building and rang the doorbell. I was greeted by Professor Buccella who walked me through some safety information and gave me goggles and my lab book. Then the fun began. After only being in the lab for about an hour, I began working with Sarina (a graduate student) and Brismar (a visiting undergraduate student) to make PIPES buffer.  It seemed easy enough, we weighed the necessary amount of PIPES and KCL, and quantitatively transferred them into a beaker, filled it to 750mL of water and began stirred the solution. It turned out there’s a lot more to making this buffer than just mixing two compounds in water and the buffer magically appears. Not only did we have to add KOH until the solution was clear and at a pH of 7, we had to che-lex it too. To “che-lex” is basically purifying the buffer, since it was going to be used for spectroscopy. Particularly, we wanted to filter out any metals, since we would use it to test metal sensors, and near exact concentrations of metals would be needed, so any unknown metals would affect the data. Hours later we finally finished making our buffer. The buffer was sacred that when we had to sacrifice a little during the process of making it, at particular sadness filled our hearts.

Making PIPES Buffer

The second day was much different than the first. Everyone got together and it was time for some lab clean-up! I’m a bit of a neat freak, so I was pretty excited. We had to clean tons of glassware, which involves a lot more than soap, water and a sponge. We re-organized cabinets and draws and at the end, and then labeled all of them (my favorite part). Some people would hate this but for me it was a learning experience, now I know where everything is!

The third day we were back to research. BB (Brismar) and I began our first titration experiment using her compound (sensor) she had made a weak prior. Before starting, we had to prepare a solution of the compound. It was pretty cool, because the we got to make aliquots of the solution then use liquid nitrogen to flash freeze them, in order to preserve the sensors in the -20o C freezer. Next we had to make a solution of MgCl2 mixed with some of the sensor, which was also fun, but we didn't get to use liquid nitrogen again. Finally we began our titrations using the fluorimeter to measure fluorescence as the concentration of magnesium was increased in the cuvette containing the solution of sensor. After collecting the data, we had to analyze it which took another day. Part of analytical chemistry is repeating the same experiment with no changes, and seeing if your results match, so we did the same titration again.

The last day of the week ended with some data analysis and a presentation from a prospective post-doc on about her doctoral work in an organic chemistry lab dealing with another type of sensor. Everyone in the lab has been so welcoming! They're always teaching me about chemistry, even if it doesn't apply to what were doing. I haven't met a group of people more passionate about what they're doing than this group. To sum the week up, not only have chemistry and I been reunited, but I’m learning chemistry has many forms besides AP Chem!