SDS-PAGE Electrophoresis Overview

Introduction

• SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) is a biochemistry technique used to separate proteins based on size (length).
• It is a common experiment in biochemistry labs and essential for protein study.
• The lecturer has extensive experience with SDS-PAGE, having run over 738 gels.

Components and Theory

• SDS: A detergent with a negative charge, hydrophobic tail, and hydrophilic head.
  • Dissociates in solution, giving a negatively charged dodecyl sulfate ion and sodium ion.
• Polyacrylamide Gel: Long acrylamide chains cross-linked to form a mesh.
  • Degree of cross-linking affects mesh tightness, important for separating protein sizes.
• Electrophoresis: Utilizes an electric gradient to move negatively charged proteins through the gel.
  • Proteins move from negative to positive charge; speed varies by protein length due to the mesh.

Protein Denaturation

• Proteins are denatured (unfolded) to ensure consistent travel through the gel, eliminating shape as a factor.
• Denaturation achieved using SDS, which coats proteins, aided by heat.
  • SDS keeps proteins negatively charged and soluble during unfolding.

Setting Up SDS-PAGE

• Gels can be homemade or precast. Homemade gels allow customization in thickness and well number.
• The gel casting involves assembling glass plates and pouring gel for polymerization.
• Sample preparation involves mixing protein samples with SDS loading buffer and heating.
  • Importance of labeling and using correct buffer concentrations highlighted.
• Use of gel loading tips to ensure accurate sample loading into wells.

Running the Gel

• Gels are placed in a buffer chamber with a charge gradient (negative top, positive bottom).
• Importance of proper setup to prevent leaks and ensure smooth operation.
• Loading involves carefully pipetting samples and molecular weight marker (ladder) into wells.

Staining and Visualization

• Post-run, gels are stained (e.g., with Coomassie Brilliant Blue) to visualize protein bands.
• Staining provides visual representation of protein purity and size comparison using a ladder.
• Tips for optimizing visualization, like using a lightbox and destaining for contrast improvement.

Practical Considerations

• Use of lab equipment, such as power boxes and gel casters.
• Handling of samples, buffers, and gels to prevent contamination or errors.
• Recognizing common issues, like buffer leakage or improper gel polymerization.

Conclusion

• SDS-PAGE is a foundational technique in biochemistry, useful for protein analysis and purification.
• Emphasized importance of proper technique, setup, and safety measures.
• Encouraged hands-on practice and familiarity with equipment for successful gel runs.

These notes provide a comprehensive overview of SDS-PAGE electrophoresis, covering both theoretical understanding and practical setup and execution. They aim to assist students in mastering this essential laboratory technique.