Beta Galactosidase Review Questions

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Email:

Instructor:

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Fill in the answers to the questions below and submit a copy of this form before attending lab.

1. A homotetramer:


2. How many molecules of lactose are associated with each chain of beta galactosidase?


3. What does a hydrolysis reaction do?


4. Why is ONPG used in the assay for beta galactosidase activity?


5. How many molecules of ONPG are associated with each chain of beta galactosidase?

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Standard Curves in the ONP Assay

6. Why are standard curves important?

Constructing a Standard Curve of the relationship between ONP concentration and absorbance

Open Minitab. Copy (ctrl C) and paste (ctrl V) the data below into Minitab (one column at a time). These data were collected by Biology 110 students in 2008.

I. Create a standard curve for the linear relationship between absorbance and concentration. See what statistic should I use to determine the appropriate statistical test for evaluating this relationship. The appropriate statistical test will graph the data, add a best fit line, and give you the equation for the line.

When setting up the analysis the parameter you can directly measure (predictor) goes on the x-axis and the parameter you want to determine goes on the y-axis. After creating your graph save it but do not close the Minitab program. Record the regression equation in your notebook. Return to this page and continue.

ONPmM
0.000
0.025
0.050
0.075
0.100
0.125
0.150
0.200
0.250		 ABS420
0.0002
0.0488
0.0992
0.1568
0.1984
0.2541
0.3124
0.3985
0.5097


7. How does the concentration of ONP in the solution change as the absorbance increases?

8. When preparing the standard curve all of the ONP solutions were diluted 1:1 with Na2CO3. How does diluting 1:1 with Na2CO3 alter the concentration and absorbance of the resulting solution compared to the non-diluted solution? We will discuss the importance of this in class as 1:1 dilution with Na2CO3 is used to stop the reaction by shifting the pH.

II. Assume that you have mixed ONPG and beta-galactosidase in a test tube with the appropriate buffer to maintain the pH. The ONPG in the solution will hydrolyze into ONP and galactose. Every minute you stop the reaction by taking 2ml of the reacting mixture and placing it in a testtube with 2ml of Na2CO3 to stop the reaction. You then measure the absorbance.

Copy and paste the data from this experiment into Minitab (one column at a time). Use the regression equation calculated from the standard curve to calculate the concentration of ONP. Graph the relationship between time and ONP (mM) concentration. Save your graph.

Time
1
2
3
4
5
6
7
8
9
10		 Abs420nm
0.044
0.104
0.143
0.212
0.247
0.297
0.352
0.405
0.435
0.479

Edit your standard curve and time graphs in Minitab. Copy and paste them into an email that will be sent to your instructor using the links provided below. Before sending the email enter the subject heading "graphs" to your email. Add captions to your graphs if this was not done in Minitab.

Warning: Send the figures in text and HTML format if that option is available on your browser. If not, copy the graphs into a word file that you attach to your email. Save a copy of your Minitab project for use in class.

mailto: edwardsr@kenyon.edu mailto: heithausp@kenyon.edu mailto: smithje@kenyon.edu
mailto: powellw@kenyon.edu  

During class you will explore the potential interaction of temperature and pH on the rate of ONPG hydrolysis. Examine the figures below, answer the final two questions, and submit this tutorial.

9. What is the optimum temperature for the reaction? 10. Between what 2 pHs does the optimum pH fall?

Please read through the lab before coming to class. We will begin by designing an experiment to which the entire class will contribute data.