Purpose
Last week you began the process of characterizing tyrosinase from Agaricus bisporus, the common white button mushroom. However, your crude extract is just that, crude. It not only contains the tyrosinase isozymes but all of the soluble protein from the mushroom. When you look at the compiled tyrosinase activity data on the Main Page, you should see differences in activity for each group. Currently, it is unclear whether these discrepancies are due to differences in extraction efficiency or actual tyrosinase concentration. Maybe the gills were ground in the mortar and pestle for longer than the skin. Maybe a stem group used less mass and prepared a more dilute extract. Since such variations in extraction procedure could lead to stark differences in measured activity, we will normalize the activity as U per mg of extracted protein. This is known as specific activity,
To do this we must first measure the protein concentration of each extract. As is often the case in research, you do not know what is the best method to use. To determine the best method, we will compare the four most common methods:
- Bradford assay
- Lowry assay
- BCA assay
- UV-Spectroscopy
Each method has its own advantages and limitations. Next week you will apply reasoning to decide as a class which method provided the most reliable data for your extracts.
Objectives
- Apply a given procedure to the preparation standards and samples for reaction-based assays
- Use a UV-Vis spectrophotometer to measure absorbance
- Construct meaning from data collected by plotting a standard curve and determining a protein concentration from it
- Evaluate the precision of different protein concentration methods
- Evaluate the accuracy of different protein concentration methods
- Create your own criteria for selecting the best protein concentration method for your application
Pre-lab preparation
Read the assigned reading (including this lesson) completely
Complete the prelab assignment at least 24 hours before lab begins
Outline the purpose, objectives, and procedures sections in your notebook. Write out the tables for each assay.