Notes on SDS-PAGE Lecture

Overview of SDS-PAGE

• SDS-PAGE: Sodium Dodicil Sulfate Polyacrylamide Gel Electrophoresis
• Technique used for protein separation based on molecular weight.
• Suitable for analyzing samples with a variety of proteins.

Mechanism of Action

• Protein Charges: Each protein has distinct charges due to amino acid side chains.
• Protein Structure: Proteins are folded due to:
  • Polarity of side chains
  • Non-covalent or covalent interactions

Denaturation with SDS

• SDS (Anionic Detergent): Denatures proteins by disrupting non-covalent bonds, leading to unfolding.
• Binding: SDS molecules bind to amino acid residues:
  • Results in similar mass-to-charge ratio for proteins.
• Purpose: Allows separation based solely on molecular weight.

Role of Beta-Mercaptoethanol

• Purpose: Reduces disulfide bonds that SDS cannot denature.
• Ensures all proteins are unfolded and have similar mass-to-charge ratios before separation.

Gel Structure and Separation Process

• Gel Composition: Polyacrylamide gel divided into two layers:
  1. Stacking Gel: Low pH; concentrates proteins into one band.
  2. Resolving Gel: Allows separation according to molecular weight.
• Electrophoresis Setup:
  • Gel placed in a chamber filled with buffer solution.
  • Protein samples pipetted into wells.
  • Electrodes connected to a power supply.

Process of Separation

• Electric Voltage Applied: Negatively charged proteins migrate from cathode to anode.
• Velocity of Proteins:
  • Large proteins travel slower.
  • Small proteins travel faster.
• Gel Structure: Acts like a net or sieve, permitting smaller particles to pass easily while larger ones take more time.

Visualization of Protein Bands

• Staining Process: After running the gel, proteins must be stained.
  • Coomassie Blue: A dye used to visualize protein bands.
• Marker Proteins: Usually loaded in the first well;
  • Known sizes used as references to determine molecular weight of sample proteins.

Western Blotting

• For identification or confirmation of specific proteins:
  • This technique is based on antibody detection.

Conclusion

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