Lecture 3 (SDS PAGE)
Principles and Techniques of SDS-PAGE (LECTURE 3)
Lecturer Information
Name: Farah Haziqah Meor Termizi, PhD
Institution: School of Biological Sciences, USM
Contact: farahhaziqah@usm.my 012-2002948
What is Protein Electrophoresis?
Definition: Electrophoresis is a technique for separating molecules.
Negatively Charged Nucleic Acids: DNA and RNA are negatively charged due to their sugar-phosphate backbone; they migrate towards the positive electrode.
Proteins: Proteins can be positively, negatively charged, or neutral, depending on their amino acid sequence and the pH of the environment.
SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis)
Overview
Purpose: Technique used for separating proteins based on their molecular weights.
Molecular Weight: Refers to the sum of the masses of all constituent amino acids in a protein.
Components
SDS (Sodium Dodecyl Sulfate): A detergent that confers a negative charge to proteins, allowing separation based primarily on size.
PAGE: Short for Polyacrylamide Gel Electrophoresis, a separation method utilizing a gel matrix.
Applications of SDS-PAGE
Protein Purification: Assessing the purity of protein samples.
Molecular Weight Estimation: Determining the molecular weight of proteins.
Protein Identification: Identifying proteins in complex mixtures.
Comparative Analysis: Analyzing protein expression levels under various conditions.
Denaturation of Proteins
Importance of Denaturation
Need for Denaturation: Proteins with different shapes/sizes must be denatured (using SDS) to lose complex structures (secondary, tertiary, quaternary).
SDS Effect: Proteins bound with SDS are negatively charged, facilitating migration towards the anode in an electric field, allowing size-based separation.
Characteristics of SDS-PAGE
Charge Variability: Protein charge varies with amino acid composition. Denaturing proteins ensures size estimation accuracy.
Gel Properties: Smaller pores in polyacrylamide gels provide better resolution for protein separation compared to agarose gels.
Preparation for SDS-PAGE
Denaturing Process
Heat Treatment: Samples heated to ~95°C breaks hydrogen bonds, linearizing proteins for consistent mass-to-charge ratios.
Reducing Agents: Compounds like β-mercaptoethanol cleave disulfide bonds aiding protein linearization.
Required Materials
Power Supplies: Convert AC to DC current.
Gels: Precast or laboratory-prepared polyacrylamide gels.
Electrophoresis Chambers: Designed to hold SDS-PAGE gels.
Buffers: Running buffer necessary for current flow and maintaining pH during electrophoresis.
Gel Casting Procedure
Stacking and Resolving Gels
Casting: The bottom layer, resolving gel (~10% acrylamide), is poured and covered with isopropanol to prevent air bubbles.
Stacking Gel: Poured on top with a lower acrylamide percentage (~4%), creating wells using a comb.
Running the SDS-PAGE
Buffer Composition
Running Buffer Functions: Provides ions for current flow and maintains gel pH (around 6.8 for stacking, 8.8 for resolving).
Sample Loading
SDS-PAGE Loading Buffer: Contains SDS, reducing agents (BME), glycerol, and tracking dye (bromophenol blue) for visibility during electrophoresis.
Protein Ladder
Purpose: A reference for estimating molecular sizes of proteins by allowing comparison of migration distance.
Visualization Techniques
Gel Staining Methods
Coomassie Blue Staining: Common method for visualizing protein bands, allowing detection of micromolar quantities of proteins.
Silver Staining: More sensitive than Coomassie, detecting 2-5 ng of protein per band.
Steps for Staining with Coomassie Blue
Gel fixed in Gel Fix solution post-electrophoresis.
Incubate in Coomassie solution for 2-4 hours.
Wash and destain the gel to highlight protein bands.
Interpretation of Results
Reading Gels: Analyzing the positions of stained bands against the protein ladder for molecular weights and comparison between samples.
Safety Precautions
Acrylamide Handling: Known neurotoxin; appropriate protective measures should be taken during preparation.