Week of 11/28/16

This week, Yi and I a pretty hands-on lab day. We performed out mini experiment involving different solutions of protein, urea, and buffer to measure how concentration of urea affects protein folding. After combining a solution of buffer and protein with another solution of buffer and urea, we used advanced spectroscopy to measure the intensity of proteins in the folded versus unfolded proteins at different concentrations.

I had to make a table of expected molarities, prepare the two different solutions, and combine the solutions in the appropriate ratios to perform the spectroscopy. Making the table of expected molarities was pretty simple, as Yi told me what the total solution volume should be (120 microliters) and what the expected urea concentration was. From there, all it took were a few molar conversions to figure out how many microliters of each solution were necessary. Below I have recorded the table of expected molarities.

Concentration (M)	Volume of protein + urea (µL)	Volume of protein + buffer (µL)
0	0	120
.2	6	114
.5	15	105
.5	24	96
1	30	90
1.2	36	84
1.4	42	78
1.7	51	69
2	60	60
2.3	69	51
2.5	75	45
2.7	81	39
3	90	30
3.3	99	21
3.6	108	12
4	120	0

Once the table was finished, we had to prepare the solutions. I used a scale to measure the proper amounts of urea to add to the protein solution, using molar mass to determine the number of grams of urea necessary for creating the proper concentration of solution. We then mixed the predetermined volumes of urea/protein solution with the buffer/protein solution to make the necessary concentration in twenty different vials. It should be noted that the amount of protein was constant in each vial, because our independent variable was urea concentration, not protein concentration.

We then place the vials of varying solution into the spectrometer to get a measure of protein absorbance under different urea concentrations (and thus proteins in different folding states). It took awhile for us to get results for our mini experiment because the spectrometer had to be warmed up and calibrated for the light intensity we were using. Unfortunately, I could not download and print the results of our experiment, but I can say that the peak absorbance lay within the middle range of urea concentration, after the pH was too basic but before it became too acidic and the protein denatured.

Although our experiment was brief and not very official,it was great practice in the lab and gave a good overview of the equipment and techniques we will be using in the future!