BIOL201 Lab W03 Biological Macromolecules and BSA Standard Curve

BIOL 201   Week 3: How do the components of biological molecules lead to their properties?     Lab: Biological Molecules     Completing and revising this activity should improve your proficiency with the following Learning Outcomes:  1. EO4  Relate roles of biological molecules to the relevant chemical properties of those molecules  2. EO4.1  Explain the properties of biological molecules using chemical principles.  3. EO4.2  Describe the relevant properties of the biomacromolecules.   EO4.3  Compare the four biomacromolecules based on their chemical composition, chemical properties, and general functions.    Part 1. SUPP Research Project – Day 2 On Day 2 of the SUPP Research Project Protocol, we will be creating extracts for testing. This is similar to the process that biomedical and biotech researchers utilize in drug discovery. 1. Collect your team’s foil packages of dried material from Day 1 and grind the material into a fine powder. 2. Transfer 1 g of dry, ground material to a labeled 1.5mL microcentrifuge tube and add 1mL methanol. 3. Cap the tube tightly and vortex for 1 minute, steep for 15 minutes, vortex for 15 seconds, steep for a final 15 minutes. 4. Centrifuge the microcentrifuge tubes at maximum speed for 1 minute. Be careful not to shake up the tube when you remove it from the centrifuge and while you are collecting the supernatant. 5. Carefully collect the supernatant (liquid portion) into a new, labeled 1.5mL microcentrifuge tube. This is the sample extract. 6. Save the extracts for next week’s experiment as instructed. 7. Use the space below to: a. Record any interesting observations from this process. b. Make note of codes or abbreviations you used for your sample extract labels. Part 2. Review of biomacromolecules Living organisms are composed of chemical elements—mostly carbon, oxygen, nitrogen, and hydrogen (together accounting for 95% of mass) along with smaller amounts of other elements. Atoms of these elements combine to form molecules. There are four main classes of large molecules (macromolecules) in living things—proteins, nucleic acids, carbohydrates, and lipids. All these classes of large molecule 1 BIOL 201 Week 3 Lab -

are organic molecules, meaning they are based on a carbon skeleton to which various “functional groups ” are attached. These functional groups include: Amino Carbonyl Carboxyl Hydroxyl Sulfhydryl Phosphate R—NH 2 R—C—R || O Ketone R—C—H || O Aldehyde R—C—OH || O R—OH R—SH O || R—O—P—O - | O - Note the 2 extra electrons shown by the negative charges on the oxygens. The “R” represents the rest of the molecule. These functional groups are parts of small stable molecules (sugars, amino acids, nucleotides) which combine (polymerize) to make large complex molecules (polymers) such as carbohydrates, proteins, and DNA. Part 3. Identifying biomacromolecules Caution: Some of the chemicals used in lab can injure skin and eyes. Please use extreme care with these chemicals and rinse with lots of water if you accidentally contact them. Testing for the presence of starch in solutions Iodine (I 2 ) dissolved in potassium iodide (KI) solution interacts with starch, a coiled polymer of glucose, and turns dark blue-black from its original yellow-brown color. So, a positive reaction is dark blue-black, and a negative reaction is a yellow-brown. It does not react with other carbohydrates that are not coiled. Therefore, this is a positive test for the presence of starch. Before you begin this test, predict the outcome of the test for each tube. To perform an Iodine Test for starch: 1. Add 1 ml of each test solution to the appropriate test tube. 2. Add three drops of the I 2 KI solution to each tube. 3. Mix gently by swirling and record the results. Iodine test for starch Tub e # Test solution Predict -/+ Actual -/+ Describe what you observed. 1 Distilled water 2 Potato blended in water 3 5% sucrose solution 4 1% starch solution 2 BIOL 201 Week 3 Lab -

Part 4. Quantifying biomacromolecules 1. Last week you created a protocol to build a dilution series using blue dye to test the limits of the spectrophotometer. This week, you will use similar steps to create a tool called a standard curve. This standard curve tool can be used to find the concentration of an unknown sample based on the mathematical relationship between the known (standard) concentrations and the observed absorbances of your dilution series tubes. In other words, the equation of the line that you generate from your data can be used to solve for the unknown sample concentration. 2. Rather than using blue dye, you will use a stock solution of protein with a known concentration – 2 mg/mL. Use the space below to organize your calculations given the following specifics: a. Each tube must contain 100  L of a diluted solution of BSA (bovine serum albumin). b. The diluted solution should be a range of concentrations (different dilutions) based on the stock solution. c. Each tube must also contain 2mL of Coomassie blue (Bradford reagent). 3. Cover each tube with Parafilm and mix by inverting slowly several times. 4. This space is available for you to record your observations . As with the lab last week, use a format that is optimal to keep your data organized and easily accessible. Once you have collected your data in the space below, use Excel to generate a graph and find the line of best fit for your data. For the standard curve, you should only use the linear portion of the data. 5. What is the concentration of your unknown sample? Show your calculations: 4 BIOL 201 Week 3 Lab -

Part 5. Weekly Synthesis. 1. In today’s lab you explored two different tests for identifying the presence of biomacromolecules and then you modified one of those tests to quantitatively determine the amount (concentration) of one of those biomacromolecules. Build a protocol to quantitatively determine the amount of carbohydrate in an unknown sample. What types of samples might serve as appropriate controls in your protocol? 2. Could you use your protocol to determine the concentration of nucleic acid in an unknown sample? Why or why not? 3. What is one thing you could modify in your protocol from Part 4 to give you more confidence in the calculated concentration of protein in an unknown sample. 4. What questions do you still have about this material? Is there a topic that you are confused on and would like more clarification? We will discuss these questions in the next lab. 5 BIOL 201 Week 3 Lab -