BIOL201 Lab W03 Biological Macromolecules and BSA Standard Curve

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Feb 20, 2024

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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 -
  List negative and positive control(s) Testing for the presence of proteins in solutions: Coomassie Blue Test (also known as the Bradford Assay) for Protein Proteins are large polymers of amino acids. The amino acids are joined by peptide bonds (a peptide is a short chain of amino acids), in which the amino group of one amino acid is joined to the hydroxyl part of the acid (carboxyl) group on another, with the loss of a water molecule. There are 20 common kinds of amino acid in living organisms, with differing functional groups (denoted R below) on the other portions of the molecule. Coomassie Brilliant Blue G-250 binds to specific amino acids, including basic and aromatic amino acids. Dye binding to amino acids causes the color of the solution to change from brown to blue. So, a positive reaction is blue and a negative reaction is brown. Again, before you begin this test, predict the outcome of the test for each tube. To perform a Coomassie Blue Test for protein: 1. Add 2 ml of each test solution to the appropriate test tube. 2. Add 1 ml of the Coomassie Blue reagent to each tube. 3. Mix gently by capping the tube with a piece of parafilm & inverting, then record the results.   Protein test using Coomassie Blue reagent. Tube # Test Solution Predict -/+ Actual -/+ Describe what you observed. 1 Distilled water 2 Whole milk 3 Potato blended in water a. Which solutions are the positive control(s) and which are the negative control(s)? You’ve now identified substances that contain starch and/or protein, but how much starch or protein is in each solution? For Part 4 of this activity, you will creating a standard curve to use as a tool to determine the concentration of protein in an unknown solution. 3 BIOL 201 Week 3 Lab -
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