LAB 3 Macromolecules

Laboratory Preparation
Pre-Lab Requirements

  • Complete macromolecules lectures.

  • Finish Week 3 pre-lab activities on Moodle.

  • Review pre-lab safety actions and safety data sheet for Oil Red O.

Objectives

  • Master pipetting technique to ensure precision and minimize contamination.

  • Execute macromolecule assays and interpret data for identifying specific macromolecules.

  • Understand positive vs. negative controls in experimental design.

Pipetting Activity

  • Instructor demonstration of pipetting: volume reading, pipettor handling, accurate transfer sequence.

  • Game: Connect 4 using microtiter plates and colored water for practical pipetting practice.

Macromolecule Assays Introduction

  • Macromolecules: Essential large molecules formed from monomers. Includes:

    • Proteins

    • Nucleic Acids

    • Carbohydrates

    • Lipids

  • Four assays to test for macromolecules:

    1. Biuret Test (Protein)

    2. Benedict's Test (Simple Carbohydrates)

    3. Lugol’s Test (Complex Carbohydrates)

    4. Sudan Red Test (Lipids)

Assays Details
1. Biuret Test

  • Detects protein presence via color change (blue to purple).

    • Positive Control: Albumin (Purple)

    • Negative Control: Distilled water (Blue)

  • Process: Mix samples with Biuret reagent; purple indicates protein.

2. Benedict's Test

  • Detects reducing sugars through color change upon heating.

    • Positive Control: Glucose solution (Orange-red)

    • Negative Control: Distilled water (Blue)

  • Process: Heat samples with Benedict's reagent; the color indicates sugar level.

3. Lugol’s Test

  • Identifies starch; positive results yield blue-black color.

    • Positive Control: 1% starch solution (Blue-black)

    • Negative Control: Distilled water (Brown)

  • Process: Mix with iodine solution; indicator of starch presence.

4. Sudan Red Test (Oil Red O)

  • Detects presence of lipids through staining.

    • Positive Control: Vegetable oil (Dark red)

    • Negative Control: Distilled water (No coloration)

  • Procedure: Apply Oil Red O, observe for dark red staining area.

Identifying Unknown Milk Samples

  • Four types of milk to identify: skim, full cream, cream, rice. Choose assays based on expected macromolecule presence:

    • Protein: Biuret Test

    • Carbohydrates: Benedict's and Lugol's Tests

    • Lipids: Sudan Red Test

Importance of Controls

  • Positive and negative controls are essential for validating the presence of macromolecules by providing a standard for comparison.

    • Example for Lugol’s: Starch (Blue-black, pos), Distilled water (Brown, neg)

    • Example for Oil Red Test: Vegetable oil (Dark red, pos), Distilled water (No coloration, neg)

Summary of Predictions for Tests

  • Milk Type | Protein Test (Biuret) | Carbohydrate Tests | Lipid Test (Oil Red O)

  • Cream: Purple, Yes | Orange-red, Yes | Red, Yes (high)

  • Full Cream: Purple, Yes | Orange-red, Yes | Moderate

  • Skim: Purple, Yes | Yellow, Yes | Low

  • Rice: Blue, No | Green, Traceable | Clear, No

Results and Identification

  • Use assay results to conclude the type of milk based on expected macromolecule presence and control comparisons.