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.