Experiment 2:
Fixed time assay b-Galactosidase
The standard assay as outlined in the lab manual should be performed and repeated as many times as necessary to achieve reproducible results. Use the Hitachi spectrophotometers. Be sure to set the correct wavelength. This will perfect your pipeting skills as well as the timing and addition of various reagents to the reaction mixture. Be sure to mix thoroughly after adding each reagent.
Determination b-Galactosidase concentration
Perform the Bradford assay on the b-galactosidase solution and use the BPA standard solution to confirm the standard curve. Use the Hitachi spectrophotometers. Be sure to set the correct wavelength.
From the enzyme activity and the protein concentration - calculate the specific activity of the b-galactosidase.
Monitor the b-Galactosidase assay as a function of time and enzyme concentration
The standard assay is conducted (at pH 8.0 as explained in the lab manual) to determine the linearity of the assay as a function of time and as a function of enzyme concentration. Use the chart recorder to obtain a continuous plot of the reaction as a function of time at two different enzyme concentrations.