BL 4820 Biochemical Techniques -- Lecture 2 - Characterizing an Acid Phosphatase
5. Optimizing the Phosphatase Assay
You will do 2 optimization procedures in the lab this week. First, you will find the optimum amount of enzyme to use in the assay. This procedure will be a little different than that described in the lab manual text since we will not be using a water bath. Here is an example of what you results may look like this week:
This plot illustrates results like those you should find in the lab this week. Since we will not be using a heated water bath for the phosphatase assay, we will use different concentrations of enzyme than are described in the lab manual text. A dilution of 1/6 of the phosphatase stock solution is shown here as being linear, while the 1/2 dilution is not. How do you make a 1/6th dilution? Take 1 part of stock phosphatase solution and add to it 5 parts of buffer (called 1:5 dilution).
Once you find the proper enzyme dilution to use for the phosphatase assay, then you will do a pH optimum. I will not illustrate it here. But if you go back to Part 3 of this lecture you will see what a typical enzyme pH optimum plot looks like.