Lab 2 Assignment: Calculations
TO BE HANDED IN AT THE BEGINNING OF LAB 3 — 10 POINTS
Be sure to include all formulas and show ALL work. Enzyme Specific Activity or Velocity contains sample calculations for determining specific activity. The introduction to this lab goes over how to calculate % yield and purification factor.
- Calculate the total protein concentration (mg/ml) in the crude extract and purified fraction. Include the equation for the linear regression line from your total protein assay standard curve. Remember the concentration you obtain from using the formula from the linear regression line does not take into account the 1:5 dilution of the PF and CE.
- Calculate the total amount of protein (mg) in the crude extract and the purified fraction by multiplying the protein concentration times the volume. For your crude extract volume, use the volume in the 15 ml conical tube after you separated the supernatant in the Corex tube from the cell debris. Assume the volume of the purified fraction to be 0.5 ml, or better, use the exact volume if you recorded it.
- Calculate the β-galactosidase activity (ONP produced per mg protein) as specific activity in one dilution of the crude extract and in one dilution of the purified fractions. Select the dilution that gives an absorbance value that is in the midpoint of the reliable range (closest to 0.5A). Units will be in μmols ONP produced/min/mg protein. Use a molar extinction coefficient of 4800M-1 cm-1 for ONP and a path length of 1 cm for the cuvettes. The appendix contains a sample calculation that should help you to do these calculations and make the unit conversions.
- Calculate the total activity (μmols ONP/min) for the crude extract and purified fractions.
- Calculate both the % yield and purification factor achieved. See the formulas in the background information for help with these calculations.
- Explain what % yield & purification factor mean in assessing the goals of a purification process.