Week of 12/5/16

This was my last internship meeting of 2016! Today was another hands-on day, so it was a lot of fun. Yi and I continued our use of the fluorescence spectrometer to determine the intensity (measured by absorbance) of folded versus unfolded proteins at various urea concentrations. To begin, I had to calculate the number of milliliters of buffer to add to the 1.1 grams of peptide.

9.7 milligrams of peptide - molecular weight = 2.9 kDa = 2900 grams per mole

9.7 * 10^-3 (1 mol / 2.9 * 10^3) = 3.34 * 10^-6 moles of protein

Final molarity should be 70 * 10^-6 M

(3.34 * 10^-6) / x = 70 * 10^-6 M

x = .048 L = 48 mL of buffer necessary

Following our calculations, we prepared our sample of protein and buffer, adding a predetermined concentrations of buffer and urea (of the sample prepared the previous week). The combined sample was pipetted into small cuvettes and placed into the large fluorescence spectrometer.

I should note that preparing the fluorescence spectrometer was no small task. Yi told me that the pressure had to be moderated very carefully, using a pump to add or remove water from the inside of the system. Traditionally, the user will turn a wheel using their hands to pump in more water, but two weeks before my visit, RPI had switched to a computer modulated pump system. Unfortunately, Yi was unfamiliar with the new computer system. We asked several other grad students in the lab for assistance, but no one could figure out why the system was malfunctioning!

Fluorescence spectrometer diagram. Image taken from: http://www.mpip-mainz.mpg.de/62430/Fluorescence_Correlation_Spectroscopy

Because of our computer troubles, we could not finish our experiment. Yi had to head to his chemistry seminar and I had to catch the shuttle back to Emma Willard. Although our experiment time was cut short, we had a lot of fun and I learned a lot about how fluorescence spectrometers work, and how delicate they are. I'm very excited for my first internship meeting of 2017!