Assays (1)

Assays (1) - ASSAYS(Activity and Bradford and Kinetics...

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ASSAYS (Activity and Bradford and Kinetics Assays) Duration: 1 week, 2 lab sessions for assays 1 week, 2 lab sessions for Kinetic assays This protocol is to guide you through two important assays that we use to characterize proteins. The first is a Bradford assay that measures protein concentrations. The second is to measure the activity of the proteins. The Bradford assay is generally applicable to many proteins, while the LacZ assay described below is more specifically tailored for the b- galactosidase a c t i v i t y o f L a cZ . I t i s v e r y important to think carefully about the experiments before initiation. The lectures will cover the basics of the experiments and theory. The TAs will assist in your experiment design, but it is critical for each group to be careful and work in coordination to save time and obtain meaningful results. The kinetic assay module is designed to be completely independent. Each group will need to devise its own protocols and parameters. There are four prelab assignments associated with these two modules due at the beginning of each session. 1. Bradford Assay 1. Describe the principles of the Bradford assay. 2. Describe Beer’s law and its components. 2. LacZ Activity Assay 1. Describe the principles of using ONPG in assaying b-galactosidase activity. 2. Describe in detail the equation used to convert absorbance to activity. 3. LacZ Kinetic Assay Part 1: determination of kcat and Km 1. Describe the derivation of MM rate equation, list all assumptions. 2. Describe the experimental design in detail, including choice of data points. 4. LacZ Kinetic Assay Part 2: IPTG as an inhibitor 1. Describe the difference between a competitive and noncompetitive inhibitor, both mechanistically and mathematically. 2. Describe the experimental design to determine what type of inhibitor is IPTG. Activity Assays Session 1: Bradford assay Overview: In this assay, the protein concentration will be determined. As discussed in lecture, a standard curve using proteins of known concentration must first be established. We will use Bovine Serum Albumin (BSA) as the standard. To test the validity of the standard curve once it is established, the TA will provide 2 protein samples with known concentrations. You will compare your results to that of TA’s known concentrations to validate your results. The Bradford assay procedure will be used later in the course to measure the amount of pure b- galactosidase protein obtained after column chromatography.
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This note was uploaded on 02/27/2010 for the course C Chem E taught by Professor Lasicka during the Spring '10 term at UC Davis.

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Assays (1) - ASSAYS(Activity and Bradford and Kinetics...

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