Alkaline Phoshatase

The goal of this experiment is to isolate and purify the enzymatic protein Alkaline Phosphotase from cells of the bacterium Escherichia coli. This protein is found in the periplasmic space in the cell membrane of E. coli. It is a dimeric protein with a molecular weight of 86,000. Its optimal enzymatic activity is above pH of 8, while its pI is 4.6. Alkaline Phosphotase itself is a zinc-containing enzyme that acts to catalyze the release of phosphoryl groups in biological systems. The resulting i

Concentration = protein amount / volume of protein = 32.08 mg / 50 mL = .6416 mg/mL

The next group of steps function to further purify the Alkaline Phosphotase. They consist of heat denaturation, ammonium sulfate precipitation, and ion exchange chromatography. Heat denaturation is possible because Alkaline Phosphotase is unique in that it is extremely heat stable. Thus, we are able to heat the reaction mixture to 75-80 degrees Celsius. This denatures most of the other proteins and leaves our Alkaline Phosphotase untouched. The denatured proteins are then removed by centrifugation as the cell parts were removed in the step above.

Ammonium Sulfate Precipitation and Dialysis:

Some topics in this essay:
Alkaline Phosphotase, Volume Protein, Specific Activity, Crude Prot, Ion-exchange Chromatography, Total Units, Phosphotase Lysozyme, Fraction Absorbance, Bradford Assay, Results Day, alkaline phosphotase, ammonium sulfate, heat denaturation, % yield, ammonium sulfate precipitation, / dt, stage 3, sulfate precipitation, amount protein, cell wall, stage 1, / dt =, stage 4 enzyme, stage 1 enzyme, dc / dt,