|
Note: In this chapter, two assays are described that are necessary in order to follow and document the purification of ß-galactosidase. Both of these assays are colorimetric assays that rely on a spectrophotometer, an instrument that measures the absorbance of light. If a review of the principles of spectrophotometry and colorimetric assays is necessary, see Seidman and Moore, Basic Lab Methods for Biotechnology, chapters 19-20. During the purification process for ß-galactosidase, two different assays will be used. The first is the Bradford assay (Bradford, 1976), which measures the total amount of all proteins present in a sample. The second is an assay that measures the enzymatic activity of the target protein, ß-galactosidase. The values obtained from these two assays can be used to calculate the amount of enzymatic activity in each milligram of protein. This latter value is known as the specific activity and will allow you to assess the purity of the target protein, ß-galactosidase. Using the assay for ß-galactosidase, you will also be able to calculate the yield of your target protein, which is the total amount of ß-galactosidase that you purified. Yield can be expressed in several ways; as the total units of enzyme; as a percentage of the number of units present in the starting material; or as a weight of ß-galactosidase. Colorimetric assays such as those described here are destructive to the sample. The total protein concentration can be reliably estimated using ultraviolet light absorption at 280 nm. Although not as accurate for solutions containing unknown proteins or mixtures of proteins, this method is not destructive to the sample and will be used to estimate the protein concentration of column fractions in Chapter 4. The ß-galactosidase assay. The assay we will use for ß-galactosidase activity measures the biological activity of the enzyme; in this case, cleavage of a specific bond found in a type of sugar known as a ß-galactoside. Like the Bradford assay, the ß-galactosidase assay is a colorimetric assay. In vivo, the substrate is lactose which is cleaved into glucose and galactose, both of which are colorless. For the assay, we use a substrate, ONPG, that produces a colored product when cleaved. The amount of enzyme activity is measured by the appearance of a yellow product that is produced when the substrate is cleaved. If there is activity in the sample, the solution will turn from clear to yellow as the enzyme does its job. A specific assay for the target protein is essential in order to follow the progress of the purification. Without a specific assay, there would be no point in trying to purify the target protein.
Important Tips for these AssaysAccurate and reproducible assays are critical to the project. These assays are the basis for all the work this semester since you are using them to track your protein and monitor its purity and activity. Therefore it is extremely important that you work out your methods and develop proper technique NOW, before we make the crude extract.
Dilute your protein appropriately. The assays we will be using are designed to measure a fairly narrow range of protein and enzyme concentrations so you will need to dilute your protein in order to be in the range of the assay. The proper dilutions for each assay will change as you proceed with the purification project. You will need to determine the correct dilutions largely through trial and error.
|
