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1. A homotetramer:
a. has 3 polypeptide chains
b. is composed of two rings containing carbon and oxygen
c. describes the structure of beta galactosidase
d. has identical amino acid chains
e. two of the above
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?
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.
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.
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.
Please read through the lab before coming to class. We will begin by designing an experiment
to which the entire class will contribute data.