SDS-PAGE Biochemistry Quiz

MD100 Medical Biochemistry I - Lab Exercise 3: Introduction to SDS PAGE

• Course: Medical Biochemistry I
• Lab Exercise: Introduction to SDS PAGE - Protein identification and characterization
• Semester: Fall 2024
• University: European University Cyprus
• School: School of Medicine

Objectives

• Introduction: SDS-PAGE laboratory technique and theoretical background
• Part A: Sample preparation – dilutions
• Part B: Sample loading – Gel running
• Part C: Gel staining and destaining
• Part D: Protein gel analysis – protein identification

Introduction to SDS-PAGE

• Technique used: Separating proteins based on molecular weight
• Electrophoresis: Separation of macro-molecules in an electric field
• SDS-PAGE: Uses a discontinuous polyacrylamide gel as a support
• SDS: Denatures proteins, creating uniformly negative charge; allowing separation based on size
• Reducing agents: (e.g., β-mercaptoethanol) cleave disulfide bonds and disrupt protein structure

The Principle of SDS-PAGE

• Charged molecules: Migrate to electrode with opposite charge
• Proteins with SDS: Become uniformly negatively charged, migrating toward the positive electrode
• SDS and reducing agents: Unfold proteins to a linear structure, charge proportional to chain length

Protein Ladder

• Molecular weight markers: Used to approximate the molecular weight of unknown proteins
• kDa values: Various molecular weight proteins for comparison

Protein Identification

• Albumin (66.5 kDa): Protein isolated from bovine serum
• Casein (24 kDa): Protein isolated from bovine milk

Materials/Equipment

• Gel: Prepared or precast polyacrylamide gels
• Vertical Gel Electrophoresis Chamber
• Protein Samples: Protein solutions
• Running Buffer: (Tris/Glycine/SDS)
• Staining Buffer: For staining the gel
• Destaining Buffer: For removing excess stain
• Protein Ladder: Pre-stained molecular weight standards
• Micropipettes & tips: for accurate measurements
• Laemmli Sample Buffer: Specifically designed buffer to prepare proteins.

Sample Preparation

• 10% Unknown Samples dilutions: 5%, 2.5%, & 1%
• Laemmli Buffer preparation: Tris-HCI (pH 6.8), SDS, glycerol, 2-mercaptoethanol, Bromophenol Blue
• Heat treatment: To denature proteins after adding Laemmli buffer to prevent protease degradation

Protocol

• Dilutions: Prepare 1 ml of 5%, 2.5%, & 1% dilutions using 10% solution
• Sample addition: Add 15 μL of each dilution to separate tubes
• Buffer addition: Add 5 μL of 4x Laemmli Sample Buffer to each sample
• Protein denaturation: Boil samples at 70°C for 2 minutes
• Running buffer: Add running buffer to gel chambers
• Protein ladder loading: Load 10 μL of protein ladder into the first well of the gel.
• Sample loading: Load 10 μL samples onto the gel
• Gel run (voltage): Run at 180 V for ~30 minutes
• Gel staining with Coomassie blue: Stain with Coomassie Blue in a dish
• Destaining: Remove Coomassie Blue and rinse thoroughly with deionized water
• Destain incubation: Incubate the gel in fresh destaining solution for 15 minutes, changing every 5 minutes.
• Overnight destain: Destain gel in deionized water overnight on a rocking table
• Photography: Take gel photograph

Protein Gel Analysis

• Single band: Indicates single protein/subunit
• Multiple bands: Indicates multiple proteins/subunits
• Band darkness: Represents protein concentration
• Comparing to ladder: Estimate molecular weight

Question 1

• Correct answer: C. Smaller proteins migrate more rapidly through the gel

Question 2

• Correct answer: A. Staining them with the dye