Outline for How to write a lab Report
BIOL 1500 Lab

The first thing you should do as you begin to write your lab report, is Make an Outline! Save this file and then change the text as you write your report, but keep this outline to help you. The report consists of three separate parts. Be consistent throughout the lab report.

To help you learn how to do this, I have given a sample outline. Once you have figured this out, you should use this as a template for the Enzyme lab due in a few weeks.

Title: Give the lab a very descriptive and meaningful title (You can do this last after you have a really good idea about what the entire lab was really all about)

I. Abstract
Write this summary after you have finished writing the rest of the report. Here you should summarize why you did the experiment (theory behind the work), what you found out, what these results mean and include an explanation of your results in relation to the theory being examined in the lab exercise.

II. Introduction
See grading rubric – Give an overall introduction to the work being done, relevant theories being tested, etc.
Part 1: Determining the effects of pH on the ALP (alkaline phosphatase enzyme)
Enzymes in the human body, maximal activity, effects of pH etc…
Hypotheses
Ho:
Ha:
Part 2: Determining the effect of various ALP (enzyme) concentration on catalytic rate
Hypotheses
Ho:
Ha:
Part 3: Determine the effect of temperature on catalytic rate
Hypotheses
Ho:
Ha:

III. Materials and Methods –
See grading rubric
Start with a materials statement for each part separately
Include all equations and methods for each part. Reference the enzyme worksheet.
Part 1: Determining effects of pH
Be sure to include the table for mixing instructions for pH here
Part 2: Determining effects of enzyme concentration
Include the tables for mixing instructions for concentration
Part 3: Determine the effects of temperature
Include the tables for mixing instructions for concentration

IV. Results
See grading rubric
Part 1: Determining the effects of pH on the ALP (alkaline phosphatase enzyme)
Reference appropriate tables and figures
You need to have a narrative about the results from this part of the lab and reference your tables and figures here.

Part 2: Determining the effect of varying the ALP (enzyme) concentration on catalytic rate
Reference appropriate tables and figures
You need to have a narrative about the results from this part of the lab and reference your tables and figures here.
Part 3: Determine the effect of temperature on catalytic rate
Reference appropriate tables and figures
You need to have a narrative about the results from this part of the lab and reference your tables and figures here.

V. Discussion
See grading rubric
Analysis of data and comparison with expected for each part of the experiment.
Summary of the data-Explain the science behind why you obtained the results as stated in your section above
Part 1: Determining effects of pH
Part 2: Determining effects of varying the concentration of enzymes
Part 3: Determining the effects of temperature

• Be sure to interpret data with respect to hypothesis-Did you fail to reject? or Did you reject?
• Answer any questions that were asked during or at the end of your lab worksheet exercise.
• Were your results similar to those obtained by other lab groups, why or why not?

Error analysis
• Examine your methods and materials for sources of experimental error and describe them here.
Conclusion
End by stating a clear conclusion of what you found overall.

VI. Literature Cited
APA style. See grading rubric

PROPERTIES OF ENZYMES Prepared by: Cortney Wilson, Naomi D’Alessio, Emily Schmitt Lavin, Ed Keith and the BIOL 1500 Team Overview In this lab, you will examine the effects of pH, enzyme concentration, and temperature on the catalytic rate of various enzymes. Some of these enzymes are involved in human digestion, and some are involved in clinical tests for certain diseases. Background This laboratory exercise is divided into three parts: 1. Determining the effects of pH on the ALP (alkaline phosphatase enzyme). 2. Determining the effect of enzyme concentration on catalytic rate. 3. Determining the effect of temperature on catalytic rate. Enzymes are extremely important for a variety of reasons. First of all, they are catalytic proteins that increase the rate of chemical reactions. Every chemical reaction that occurs inside the cell is catalyzed by an enzyme. Thus, life itself would probably be impossible without the participation of enzymes in living processes. Second, enzymes help us digest the food we eat. Without these enzymes, we would die from lack of nutrients and energy. Third, enzyme assays are used to diagnose a wide variety of diseases, and are therefore incredibly valuable in the practice of medicine. Enzymes are different from inorganic catalysts because their activity is affected by many factors and controlled in many ways. The control of enzyme activity can be subdivided into three different categories: (1) the actual properties of the enzyme, (2) the kinetics of the enzyme, and (3) the type of enzyme. In this experiment, you will examine the effect of pH, temperature, and enzyme concentration on the catalytic rate of the enzyme alkaline phosphatase (ALP). An enzyme’s activity is often limited to a relatively narrow pH. On either side of this range, enzymatic activity drops off rapidly, so a plot of rate verses pH frequently yields a bell-shaped curve: We call the pH at which an enzyme shows maximal activity the optimum pH. For most enzymes in the human body, the pH is near neutral (pH ? 7.0), but some enzymes have unusually high or unusually low pH optima, as you will see. Similarly, enzymes have an optimum temperature at which they show maximal activity. Most enzymes have temperature optima between 20-40ºC, although some enzymes have unusually high temperature optima and some have unusually low temperature optima. For most enzymes the rate of reaction doubles for every 10ºC increase in temperature (Q10 effect). However, this effect is limited by the denaturing of the enzyme as temperature increases. Once denatured, the enzyme cannot catalyze its reaction. The rate of an enzyme controlled reaction is also controlled by both the concentration of the enzyme and the concentration of the substrate. As the concentration of either is increased the rate of the reaction is also increased. However, if either the enzyme or the substrate becomes limited, the rate of the reaction will remain constant. In this lab you will hold the concentration of the substrate constant and you will vary the concentration of the enzyme. Human digestion occurs by a series of hydrolytic reactions, which break down large polymeric food molecules into smaller monomers that our bodies can absorb. An enzyme catalyzes each of the hydrolytic reactions. Cells synthesize macromolecules such as proteins, fats, carbohydrates, and nucleic acids through the enzyme controlled processes of synthesis. Many diseases produce elevations in the concentration of specific enzymes in the blood plasma. This can lead to cell injury and/or death caused by the disease, and the consequent release of cell contents into the circulatory system. In other cases, diseased cells overproduce certain enzymes and release them. Some released enzymes are specific for a given disease, and these “marker enzymes” can be used to diagnose the condition. For example, the enzyme alkaline phosphatase can be used to diagnose a variety of diseases such as bone cancer, jaundice and cirrhosis of the liver. Enzyme Activity pH Alkaline phosphatase (ALP) catalyzes the hydrolysis of a phosphate group attached to proteins or lipids in vivo. In this experiment, we will use an in vitro substrate, para nitrophenol-phosphate (pNPP), which is colorless. Hydrolysis of the phosphate yields para nitrophenol, which is yellow. The appearance of the yellow product will be monitored over time, and used as an index of the rate of reaction. Procedure The solutions used are the following: Solution A – Alkaline buffer Solution B – Substrate (0.003 M disodium p-nitrophenyl phosphate (dNPP)) Solution C – Enzyme ALP, low concentration Solution D – Enzyme ALP, high concentration Solution E – Solution B/dH2O. This solution will be used for the pH experiment. Solution F – Solution A/Solution B. This solution will be used for both the temperature and concentration experiments. ? Lab Report Hint: Think about the how solution E differs from solution F. Why Solution F is used for the temperature and concentration experiments BUT is not used for the pH experiment. Caution: Alkaline phosphate buffer solution and pNPP substrate are irritants. Prevent eye, skin, and clothing contact. Part One – Estimating the optimum pH for ALP activity 1. Prepare Solution E by adding 6.5mL Solution B (para nitro phenolphosphate – pNPP) with 6.5mL dH2O. 2. Label 4 cuvettes and prepare them as indicated in Table 1. Do not add solution D to the cuvettes yet. 3. Place a strip of broad-range pH paper on a clean, dry watch glass and immediately after mixing, remove a drop of each solution and place the drop on the pH paper. (ALP) Alkaline phosphatase pNPP paranitrophenol-phosphate (clear) pNP paranitrophenol (no extra phosphate) (yellow) 4. Record the pH of each solution in Table 2. Table 1. Mixing Instructions for pH Experiment Tube Relative pH Solution E 0.2 M HCl 0.1 M Na2CO3 Distilled water Solution D High conc. enzyme Total Volume 1 control — 3 mL — — 2 mL — 5 mL 2 Acidic 3 mL 1.9 mL — — 100 ?L 5 mL 3 Neutral 3 mL — — 1.9 mL 100 ?L 5 mL 4 Basic 3 mL — 1.9 mL — 100 ?L 5 mL 5. Tube 1 is your control and you should use this tube to blank the spectrophotometer at 405 nm when you start this experiment. You need to blank the spectrophotometer to set the absorbance at 0 for the clear (blank) solution. N.B. Do not add solution D (enzyme ALP high concentration) until you are ready to actually take the readings of EACH cuvette in the spectrophotometer. When you add solution D, cover with parafilm and invert carefully to mix. It is important that all solutions be well mixed or the color change will not happen throughout the tube and will not be detected by the spectrophotometer. 6. Begin this procedure with cuvette 2. Add solution D to cuvette 2. Using the parafilm mix carefully and place cuvette 2 in the spectrophotometer and record the absorbance reading immediately (time 0) and then every 30 seconds for five minutes. Record the readings in Table 2. 7. Repeat this procedure with cuvette 3 and then cuvette 4. Table 2. Effect of pH on the Activity of the Enzyme ALP Tube 2 3 4 pH Initial (0 s) 30 s 60 s 90 s 120 s 150 s 180 s 210 s 240 s 270 s 300 s Part Two – Determining the Effect of Enzyme Concentration on Catalytic Rate Part Three – Determining the Effect of Temperature on Catalytic Rate Procedures to prepare for both concentration and temperature experiments: Also refer to the mixing tables below: 1. In a 50 mL beaker, mix 15 mL of solution A with 15 mL of solution B (these are at the front bench). This is now solution F and will be used for the concentration and the temperature experiments. 2. Label all 8 of your cuvettes at the very top, 1a, 2a, 3a, 4a and 1b, 2b, 3b, 4b. 3. First, prepare the blank cuvette 1a as indicated in the mixing tables below. Use it to set the spectrophotometer to 0 at 405 nm. *Think about it: Why do we need to re-blank the spectrophotometer if we already blanked it for the pH experiment? What has changed about our blank? Then do not touch the wavelength or blank button again. 4. After blanking the spectrophotometer in this way, you will be adding the enzyme to cuvette 1 to become 1a as indicated in the table 3 below BUT don’t add the enzyme until you are ready to measure the color change in the spectrophotometer! Wait until you get to that step below. 5. Using a micropipette, add 3 mL of your mixed solution F to each of your labeled cuvettes (1a-4a and 1b – 4b). 6. For Part 3, prepare the “b” cuvettes for the temperature experiment by: Placing cuvette1b and a micro-centrifuge tube labelled 1b with 100uL of solution C in the refrigerator (4?C). Placing cuvette 2b and a micro-centrifuge tube labelled 2b with 100uL of solution C on the bench at room temperature Placing cuvette 3b and a micro-centrifuge tube labelled 3b with 100uL of solution C in a 32?C water bath. Placing cuvette 4b and a micro-centrifuge tube labelled 4b with 100uL of solution C in a 60?C water bath. 7. Do NOT begin the next step until you are ready to take data using the spectrophotometer. Be sure that someone has a watch with a second hand or a digital readout. Also, have a small square of parafilm ready to mix the content of the cuvette. Part two: Determining the Effect of Enzyme Concentration on Catalytic Rate 1. There should be 3 mL of your mixed solution F in each cuvette (1a – 4a) from step 5 above. 2. Using a micropipette, add 100 ?L of solution C (Low ALP concentration) to cuvette 1a, quickly cover with parafilm, invert to mix, remove parafilm, and place into spectrophotometer. Do this as rapidly as possible, and then begin to record the absorbance readings starting with the first reading at 0 time. Read the absorbance every 30 seconds for 5 minutes after you insert the mixed cuvette into the spectrophotometer and record your data in the appropriate table. 3. Using a micropipette, add 400 ?L of solution C (Low ALP concentration) to cuvette 2a, quickly cover with parafilm, invert to mix, remove parafilm, and place into spectrophotometer. Do this as rapidly as possible, and then begin to record the absorbance readings. Read the absorbance every 30 seconds for 5 minutes after you insert the mixed cuvette into the spectrophotometer. 4. Using a micropipette, add 200 ?L of solution D (High ALP concentration) to cuvette 3a, quickly cover with parafilm, invert to mix, remove parafilm, and place into spectrophotometer. Do this as rapidly as possible, and then begin to record the absorbance readings. Read the absorbance every 30 seconds for 5 minutes after you insert the mixed cuvette into the spectrophotometer. 5. Using a micropipette, add 500 ?L of solution D (High ALP concentration) to cuvette 4a, quickly cover with parafilm, invert to mix, remove parafilm, and place into spectrophotometer. Do this as rapidly as possible, and then begin to record the absorbance readings. Read the absorbance every 30 seconds for 5 minutes after you insert the mixed cuvette into the spectrophotometer. Part three: Determining the Effect of Temperature on Catalytic Rate 1. Retrieve cuvette 1b and the micro-centrifuge tube with solution C from the refrigerator. Make sure to work quickly so that the liquid stays cold. Using a micropipette, add the contents of the micro-centrifuge tube (solution C) to cuvette 1b. Cover quickly with parafilm, invert to mix, remove parafilm, wipe down any condensation that may have formed, and place into the spectrophotometer. Do this as rapidly as possible and begin to record the absorbance readings. Read the absorbance every 30 seconds for 5 minutes after you inserted the mixed cuvette into the spectrophotometer. 2. For cuvette 2b, add the contents of the micro-centrifuge tube (100 ?L of solution C at room temperature) to the cuvette 2b. Cover quickly with parafilm, invert to mix, remove the parafilm and place into spectrophotometer. Do this as rapidly as possible, and then begin to record the absorbance readings. Read the absorbance every 30 seconds for 5 minutes after you insert the mixed cuvette into the spectrophotometer. 3. For cuvette 3b, retrieve the cuvette and the micro- centrifuge tube from the 32?C water bath and wipe down the outside of the cuvette. Add the contents of the micro-centrifuge tube (100 ?L of solution C) to cuvette 3b. Cover quickly with parafilm, invert to mix, remove parafilm, and place into spectrophotometer. Do this as rapidly as possible, and then begin to record the absorbance readings. Read the absorbance every 30 seconds for 5 minutes after you insert the mixed cuvette into the spectrophotometer. 4. For cuvette 4b, retrieve the cuvette and the micro- centrifuge tube from the 60?C water bath and wipe down the outside of the cuvette. Add the contents of the micro-centrifuge tube (100 ?L of solution C) to cuvette 4b. Cover quickly with parafilm, invert to mix, remove parafilm, and place into spectrophotometer. Do this as rapidly as possible, and then begin to record the absorbance readings. 5. Read the absorbance every 30 seconds for 5 minutes after you insert the mixed cuvette into the spectrophotometer. Table 3. Mixing Table for Effect of Concentration on Catalytic Rate (Beware of the Dark Bold Line in the table! Note: Do not add solutions either C or D until you are ready to actually take the readings in the spectrophotometer. Add C or D to one tube at a time and stagger the tubes.) Tube Relative Enzyme Conc. Sol F (comprised of Sol A/B Buffer/substrate stock solution) Sol C Low ALP (Enzyme) conc. Sol D High ALP (Enzyme) conc. 1 blank (no enzyme) 0 3 mL — — 1a blank plus enzyme Lowest 3 mL 100 ?L — 2a Medium 3 mL 400 ?L — 3a Higher medium 3 mL — 200 ?L 4a Highest 3 mL — 500 ?L Notes: First, prepare the blank cuvette as indicated above. Use it to set the spectrophotometer to 0 at 405 nm. Then do not touch the wavelength or blank button again. Why do you have to blank the spectrophometer again if you already blanked it in the pH experiment? What is different about tube 1 in the concentration and temperature experiments compared to the pH experiment? After blanking the spectrophotometer in this way, add the enzyme to cuvette 1 as indicated in the table above and following the procedure below. Table 4. Mixing Table for Effect of Temperature on Catalytic Rate (Beware of the Dark Bold Line in the table! Note: Do not add solutions C and D until you are ready to actually take the readings in the spectrophotometer. Add C to one tube at a time. Do not remove the cuvettes from their temperature environments until you are ready to add the enzyme (solution C) to that cuvette. Also, do not add solution C until the tubes have been at the proper temperature for the indicated amount of time.) Tube Temperature (ºC) Sol F (Buffer/substrate stock solution) Sol C Low ALP (Enzyme) conc. 1b 4 3 mL 100 ?L 2b 23 3 mL 100 ?L 3b 32 3 mL 100 ?L 4b 60 3 mL 100 ?L Table 5. Data Table for effect of enzyme concentration and temperature on the rate of an enzyme controlled reaction. Record absorbance for each tube in this table and then bring your group data to the lab computer with data from all the individual groups in your lab section. Absorbance Readings for Each Cuvette concentration temperature Time (sec) 1a (low) 2a (med) 3a (med-high) 4a (high) 1b (4oC) 2b (20 oC) 3b (32 oC) 4b (60 oC) Initial 30 s 60 s 90 s 120 s 150 s 180 s 210 s 240 s 270 s 300 s Name Date LABORATORY REPORT FORM – This lab will be written up as a full lab report. Use the information below to help guide you with writing your lab report. Check your syllabus for the due date of this report. Although you will have shared data with members of your lab team, all work on your report should be done independently. Writing the lab report. – Your lab report should have the following sections: Title Abstract Introduction Materials and Method Results – including tables and graphs Discussion Literature Cited Follow the directions in the Lab Report Format handout when writing up the lab. Also, carefully read the handout, “How to write a great lab report” developed by Dr. Joshua Loomis. Consider the following when writing your report. 1. At the end of the introduction, be certain to state a Null Hypothesis and an Alternate Hypothesis for each section of the lab. 2. In the Materials and Methods section you describe the process used in this lab. Include tables 1, 3, and 4 which indicate the materials used in each section of the experiment. Make certain you include descriptive titles for each table. 3. In the Results section you should include your data tables and graphs. Include numbers and titles for each data table and each graph. There should be a minimum of six graphs in the results section 4. Figure 1 should be a scatter plot of your data on the effect of pH on the enzyme controlled reaction. Time should be your independent variable and absorption should be your dependent variable. Add a trendline for each pH. Then add the equation of each line. The equation will be in the form of Y=MX+B, where M is the rate of the reaction 5. Figure 2 should be a column graph that compares the rate of the reactions for each pH. The pH’s are your independent variable and the rates of the reactions are the dependent variables. 6. Figure 3 should be a scatter plot of your data on the effect of enzyme concentration on the enzyme controlled reaction. Time should be your independent variable and absorption should be your dependent variable. Add a trendline for each concentration. Then add the equation of each line. The equation will be in the form of Y=MX+B, where M is the rate of the reaction 7. Figure 4 should be a column graph that compares the rate of the reactions for each concentration. The concentrations are your independent variable and the rates of the reactions are the dependent variables. 8. Figure 5 should be a scatter plot of your data on the effect of temperature on an enzyme controlled reaction. Time should be your independent variable and absorption should be your dependent variable. Add a trendline for each temperature. Then add the equation of each line. The equation will be in the form of Y=MX+B, where M is the rate of the reaction 9. Figure 6 should be a column graph that compares the rate of the reactions for each temperature. The temperatures are your independent variables and the rates of the reactions are the dependent variables. 10. Include in the discussion section of your report and explanation as to why a buffer is used when testing the effect of temperature and concentration and the rate of an enzyme controlled reaction; but no buffer is used when testing the effect of pH. 11. In the literature cited section of the report you should reference all sources used. This should include not only your textbook, lab instructions, and notes but also at least one peer reviewed journal article.