Pre-Lab. 1. What do people mean when they say teenagers have raging hormones?

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1 Name: Period: Date: You ve got MALE! Hormonal Control of Male Reproductive Processes This simulation explores how sex determination works. You will learn about the impact of testosterone concentration on phenotypic changes by using a simulated ELISA to measure testosterone levels in samples taken over the course of a male s lifetime. This will provide you with the background information to consider the threats to male fertility. Introduction: The SRY gene (located on the Y chromosome) explains why males and females have different gonads, but what about the rest of the differences between male and female bodies? The answer to this question depends a lot on hormones. Hormones are chemical messengers produced by the endocrine glands that travel through your body via the bloodstream to mediate a number of essential biological processes. Some of these processes include: reproduction, growth and development, and maintaining homeostasis. The hormone we will be focusing on in this lab is the steroid hormone testosterone. Testosterone is needed for the development of many of the features that we consider part of being male. Later in this lesson you will learn how the presence of testosterone produces physical changes and you will also learn about the implications of varying quantities of testosterone throughout a male s lifespan. In this lab you will use samples from males at different points in their lifespan to measure testosterone levels, and you will try to figure out what the implications of these various testosterone levels might be. Let s think about the lab before we begin Pre-Lab 1. What do people mean when they say teenagers have raging hormones? 2. Which hormones are associated with male development? What is the purpose of this hormone? Getting ready for the lab 3. What will the results of the ELISA Essay tell you? 4. You will be testing known concentrations of testosterone (standards) along with samples taken throughout an average man s life (samples with unknown concentrations). What is the purpose of testing the standards? 5. You will be testing standards in two wells instead of just a single well. Why is it important that you test each standard twice? 1

2 2 [NUBIO MALE HORMONE LAB] 6. Predict how testosterone levels fluctuate (change) over the course of a male s lifetime by drawing the levels on the graph below. Graph 1 Prediction of Testosterone Production Levels in Different Phases of Life 7. Record how you will arrange your well plate in the diagram below. This will tell you the location of the standards and samples so you can keep track of your data. You will need two wells for each standard (20, 15, 10, 5, 2.5, 0 nmole/l) and one well for each samples (2 nd Trimester, Birth, 2 months, 1, 7, 10, 13, 16 22, 40, 60 and 80). Figure 2: Well plate set-up for an ELISA assay A B C D E F G H 2

3 You ve got MALE! Completing and analyzing an ELISA ❶ Add 100 µl of capture antibody to each well designated for your standards and twelve patient samples. Use Figure 2 to keep track of which wells have been filled with reagents by placing a mark next to each well. ❷ Incubate the plate at room temperature (let the plate sit untouched at a room temperature) for 5 minutes. 8. What is an ELISA test? While your plate is incubating, view the direct ELISA animation from 9. What do you do in an ELISA test? 10. How does an ELISA test determine concentrations of testosterone? 11. Record and label the sequence of steps performed in a direct ELISA in the diagram provided, below. Be as specific as possible. ❸ After 5 minutes, empty the contents by turning the well plate upside-down and shaking the liquid into a sink until all of the liquid has been removed. Blot the well plate on paper towel to remove any remaining liquid. ❹ Remove any unbound antibody from the wells by washing the well plate with Antibody Wash Solution. Fill wells to the top with Antibody Wash Solution Buffer. Empty the contents as above. Repeat this wash step 3 more times. ❺ Using the micropipette, add 50 µl of each standard/each patient sample to their corresponding wells designated in Figure 2. ❻ Place the plate on the designated tray to allow the plate to incubate (let the plate sit) overnight. This allows the hormone to bind to the antibodies in the plate. Your teacher will wash the well plates for you and add the secondary antibody linked enzyme. Tomorrow, you will finish the assay by adding a substrate to the wells and looking for a color change reaction to occur. 3

4 4 [NUBIO MALE HORMONE LAB] You ve got MALE! Using an ELISA to test hormone concentrations You will complete the ELISA assay by adding the substrate ABTS and observing the presence or absence of a color change. ❶ Obtain your well plate, ABTS, and a p-200 micropipet Important: Read these lab notes before moving on! Add the ABTS Substrate Solution to each standard and sample well. After adding the ABTS Substrate Solution, mix the contents of the well plate thoroughly by removing and adding the liquid back to the well plate three times to make sure you can see the color change. It is important to add the stop solution to the wells in the SAME order as you added the ABTS Substrate Solution. If the pipette tip touches liquid from the well, change tips to prevent contamination. ❷ Place your well plate on a white sheet of paper (You can put your well plate directly on Figure 2). ❸ Add 100 µl of ABTS Substrate Solution to each of the standard and patient sample wells. Keep track of the order in which you add the substrate in your well plate on Figure 2 (you want to continue using the same order each time). ❹ Incubate the plate at room temperature for approximately 5 minutes. Watch for a color change reaction. If a color change reaction does not occur within 10 minutes, ask your teacher to look at your plate. ❺ Following the same pattern as in STEP ❸, add 100 µl of the SDS Stop Solution to each well to stop the reaction and record the length of incubation time. ❻ Record the colors of the samples in your data table. ❼ Compare the color of each sample to the standards. Which standard is each patient sample closest to? Use the standards to approximate the concentration of testosterone in each sample. Record this value in your data table. ❽ Add your data to the class data table. Obtain an average for the testosterone concentration for each sample 4

5 Please Detach This Page and Turn it in! Name: Period: Date: You ve Got Male Record Sheet 1. Purpose: Explain what you will find. Make sure you include the conditions of the experiment. (2 pts) 2. Hypothesis: What do you expect to happen and why you think this will occur. Look at your prediction graph on 2. When did you think the testosterone levels would be the greatest? What did you think would happen to the levels over time? (2 pts) 3. Manipulative skills: Collecting data properly, carrying out techniques and working safely. (5 pts) 4. Data: Observations and data from the lab. Include a title explaining the condition of the lab with at least 15 words (5 pts) Patient Sample Table 2: 1 2 nd Trimester (-0.4 years) 2 Birth (0 years) 3 2 months (0.16 years) 4 1 year Age Color of Sample Other Observations Testosterone Concentration nmole/l Class Average Testosterone Concentration nmole/l 5 7 years 6 10 years 7 13 years 8 16 years 9 22 years years years years 5

6 6 [NUBIO MALE HORMONE LAB] 5. Translate your data into a graph. Include a title for your graph explaining the condition of the lab with at least 15 words (5 pts) Graph 2: 6. Claims and Evidence: Describe the overall trend of testosterone throughout male s life. (2 pts) 7. Evaluating procedure: Evaluates weaknesses and limitations of the lab. (2 pts) What did you measure? How well were you able to collect data? Identify sources of error that may lead to false positive or false negative test results. Analysis Questions: The science you need to know. (2 pts each) 8. When is testosterone concentration the highest and the lowest? Explain how this relates to what happens during this time in a male s life? 9. Compare your hypothesis with your actual results. Describe the similarities and differences between the two. Does your data support your hypothesis? Were you shocked by anything? Explain. 10. A group of students were not closely reading their lab procedure. They only added 10 microliters of ABTS to their wells. What would happen to the results of their experiment? Would they get the same results? Explain. 6