I performed an ammonium sulfate precipitation to purify my protein of interest.
ID: 483220 • Letter: I
Question
I performed an ammonium sulfate precipitation to purify my protein of interest. Following the precipitation, I had two samples to test for activity: The pellet and the supernatant. The pellet was resuspended in 4.00 mL and the supernatant volume was 7.50 mL. My data is shown below. a. Make a graph using Excel of absorbance versus time for both Sample A and Sample B using Excel and paste below. Draw a linear regression through the linear part of each curve. Determine the slope and report for each sample in units of A_340/sec. b. According to the graph you made, is the majority of my protein of interest in the pellet or supernatant? Why? A separate activity assay was performed after the G-25 column using a 1:10 dilution of protein and resulted in a slope of -0.0036 A_340/sec. Convert the slope to activity in units of U/mL.Explanation / Answer
a) The plots for the supernatant and the pellet are shown below:
Plot of A340 vs t (s) for supernatant liquid; The linear equation on the top denotes the equation for the complete set. The linear equation at the bottom denotes the linear equation for the most linear part of the plot (Series 2)
Plot of A340 vs t for pellet; The plot is more or less through all the data points – indicated by a high value of R2.
The slope for the supernatant is -0.0013 A340/s.
The slope for the pellet is -0.0013 A340/s (ans).
b) The majority of the protein of interest appears to be in the supernatant. The absorbance of a sample is directly proportional to the concentration of the sample. At t = 0, the absorbance of the supernatant is much higher than that of the pellet. This should directly indicate that the supernatant contains a higher concentration of the protein than the pellet.