Lab 9 - Isolation of Acid Phosphatase from Wheat Germ Gel...

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Isolation of Acid Phosphatase from Wheat Germ Gel Filtration and Sephadex G75 Column and Ion Exchange Chromatography 1
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Isolation of Acid Phosphatase from Wheat Germ Gel Filtration and Sephadex G75 Column and Ion Exchange Chromatography University of Texas at Austin Department of Chemistry and Biochemistry Introduction Enzyme Data and Observations When we carried out our experiments, none of the procedures deviated from the lab manual’s procedures. Table 1 : Gives the extraction and purification amounts with ammonium sulfate Ammonium Sulfate Precipitations Vol. of Cell Extract 30.0 mL Mass of (NH 4 ) 2 SO 4 – 1 st 4.916 g Vol. of 30% Supernatant 30.0 mL Mass of (NH 4 ) 2 SO 4 – 2 nd 6.410 g Vol. of 65% Supernatant 32.8 mL Table 2 : Gives the A 205 for the WGAP assay WGAP Assay Tube A 205 1 Blank 2 0.284 3 0.583 4 1.329 Upon calibrating the G75 column, tubes 9-12 that came off the column had the Blue Dextran in the samples. Each of the tubes had 1mL of the elution solution. The height of the column was 13.5 cm and the diameter was 1.8 cm. Table 3 : Gives the A 280 for the Sephadex G75 Chromatography Fractions 2
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G75 Fractions 3
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Fraction A 280 Fraction A 280 1 0.026 21 1.291 2 0.158 22 1.27 3 0.16 23 1.353 4 0.212 24 1.245 5 0.243 25 1.537 6 0.01 26 0.276 7 0.284 27 1.162 8 0.417 28 0.901 9 0.634 29 1.016 10 0.904 30 0.86 11 0.788 31 0.938 12 0.979 32 0.825 13 1.018 33 0.703 14 1.02 34 0.604 15 1.005 35 0.515 16 1.092 36 0.539 17 1.169 37 0.583 18 1.156 38 0.525 19 1.156 39 0.418 20 1.204 40 0.552 Table 4 : Gives the WGAP assay of the six highest A 405 readings of the G75 fractions
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