Bio135_assignment_1 - Name Soo-Khee Chia ID 005811923 Biol...

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Name: Soo-Khee Chia ID #: 005811923 Biol 135 Assignment #1 CALCULATION: (8-22) The purification of a protein usually requires multiple steps and often involves several types of column chromatography. A key component of any purification is an assay for the desired protein. The assay can be a band on a gel, a structure in the electron microscope, the ability to bind to another molecule, or an enzymatic activity. The purification of an enzyme is particularly instructive because the assay allows one to quantify the extent of purification at each step. Consider the purification of the enzyme shown in Table 8-1. The total volume, total protein, and total enzyme activity are shown at each step. Procedure Total Volume (mL) Total Protein (mg) Total Activity (units) Specific Activity (units/mg) Crude Extract 2,000 15,000 150,000 10 Ammonium Sulfate Precipitate 320 4,000 140,000 35 Ion-exchange Chromatography 100 550 125,000 227.27 Gel-filtration Chromatography 85 120 105,000 875 Affinity Chromatography 8 5 75,000 15,000 A.For each step in the purification procedure, calculate the specific activity of the enzyme (units of activity per mg of protein). How can you tell that purification has occurred at each step? 150,000 (units of activity) Crude Extract = --------------------------------- =10 (units/mg) 15,000 (mg of proteins) 140,000 (units of activity) Ammonium sulfate precipitation = --------------------------------- =35 (units/mg) 4,000 (mg of proteins) 125,000 (units of activity) Ion-exchange chromatography = --------------------------------- =227.3 (units/mg) 550 (mg of proteins) 105,000 (units of activity)
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Gel-filtration chromatography = --------------------------------- =875 (units/mg) 120 (mg of proteins) 75,000 (units of activity) Affinity chromatography = --------------------------------- =15,000 (units/mg) 5 (mg of proteins) If there is an increase in each of the specific activity calculated, we can tell that the purification
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Bio135_assignment_1 - Name Soo-Khee Chia ID 005811923 Biol...

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