9. Below is the data from five steps used in a protein purification. Answer the questions below. Fraction | U/mL | Total Units | % Recovery | mg/mL protein | Specific Activity (U/mg) 20,000 g supernatant | 45.5 | 1989 | 100 | 8.5 | 5.3 (NH4)2SO4 Precipitation | 225 | 1692 | 85 | 12.5 | 18 Pooled IEX Fractions | 202 | 1631 | 82 | 11.5 | 17.5 Pooled Gel Filtration Fractions | 175 | 1293 | 65 | 1.9 | 92.1 Pooled Affinity Fractions | 500 | 1012 | 51 | 10.2 | 49 a. Which step in this table appears to have the best improvement in purity? Briefly explain. 4 pts. b. Explain why the values of "U/mL" go up and down during the purification steps. 3 pts. c. Explain why the values of "Total units" decrease during successive steps. 3 pts.
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Complete the purification table. Compute yield and purification factors on a "per step" basis, not cumulative. In the end, you will calculate the overall yield and purification factor. Step | Amount of protein (mg) | Amount of activity (U) | Specific activity (U/mg) | Yield (%) | Purification factor Starting point | 5000 | 500 | | — | — after ammonium sulfate cut (dialyzed) | 1500 | 450 | | | After gel filtration column | 600 | 300 | | | After ion exchange column | 100 | 240 | | | After affinity column | 2 | 180 | | | Cumulative yield = Cumulative purification factor = Which is the best step?
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