SDS-PAGE Notes

Sodium DodecylSulphate-PolyAcrylamide Gel Electrophoresis (SDS-PAGE)

What is Electrophoresis?

  • Electrophoresis is a laboratory technique used for separating molecules based on their charge.

Separation Principle

  • Charged molecules are separated based on their electrical charge and size within a matrix.
  • This technique allows for:
    • Analysis: Identify components in a mixture of charged molecules.
    • Purification: Isolate specific charged molecules.

Gel Matrix

  • The gel matrix is composed of either agarose or polyacrylamide.
  • Polyacrylamide:
    • A cross-linked polymer of acrylamide.
    • Acrylamide is a potent neurotoxin and should be handled with care!

Polyacrylamide Gels

  • Have smaller pores than agarose gels.
  • Offer a high degree of resolving power.
  • Can separate DNA fragments ranging in size from 10-500 bp.
  • Can separate DNA fragments differing in size by even one nucleotide.
  • Also used to separate protein molecules.

Protein Electrophoresis

  • Separates proteins based on Size (Molecular Weight - MW).
  • Allows us to:
    • Characterize proteins.
    • Quantify proteins.
    • Determine the purity of a sample.
    • Compare proteins from different sources.
  • A crucial step in Western blot analysis.

Protein Considerations

  • Proteins, unlike DNA, do not have a constant size-to-charge ratio.
  • In an electric field:
    • Some proteins will migrate toward the positive pole.
    • Some will migrate toward the negative pole.
    • Some will not move at all because they are neutral.
  • Native proteins can be run on native gels.
  • Alternatively, proteins can be forced to acquire the same size-to-charge ratio using SDS.

SDS-PAGE

  • SDS-PAGE (sodium dodecylsulphate-polyacrylamide gel electrophoresis) separates proteins according to their size.
  • Understanding SDS and PAGE is crucial to understanding the method.

Sodium Dodecylsulphate (SDS)

  • The goal is to separate many different protein molecules of a variety of shapes and sizes by size alone.
  • Proteins need to be linearized to eliminate the influence of shape.
  • SDS is used to convert all proteins to the same shape.

SDS as a Detergent

  • SDS is a detergent that can dissolve hydrophobic molecules and has a negative charge (sulfate) attached.
  • When SDS is added to proteins:
    • Proteins are solubilized by the detergent.
    • Proteins become coated with many negative charges.

SDS Treatment

  • A protein sample, often freshly isolated and unpurified, is boiled in the presence of SDS.
  • The end result has two key features:
    1. All proteins contain only primary structure.
    2. All proteins have a large negative charge, which means they will all migrate toward the positive pole when placed in an electric field.
  • Proteins migrate through the gel toward the positive pole at a rate proportional to their linear size.
  • Molecular weights with respect to size markers can then be determined.

Impact of SDS on Protein Structure

  • SDS nonpolar chains arrange themselves on proteins and destroy secondary, tertiary, and quaternary structures.

SDS and Protein Charge

  • So much SDS binds to proteins that the negative charge on the SDS drowns out any net charge on protein side chains.
  • In the presence of SDS, all proteins have a uniform shape and charge per unit length.

Polyacrylamide Gel

  • The polyacrylamide gel contains tunnels of different diameters.

Protein Movement in Gel

  • Smaller proteins move through the gel faster.
  • Larger proteins move at a slower pace.

Components of the System

  • DC Power Source, Reservoir/Tank, Glass Plates, Spacers, and Combs
  • Support medium: Gel (Polyacrylamide)
  • Buffer System: High Buffer Capacity
  • Molecules to be separated: Proteins or Nucleic Acids

Vertical Gel Format

  • The system includes:
    • Reservoir/Tank
    • Power Supply
    • Glass Plates, Spacers, and Combs
  • Stacking Gel: to concentrate the proteins into a narrow band before they enter the resolving gel
  • Running Gel: Also known as the resolving gel is where the proteins are separated by size

SDS-PAGE Process

  • (A) SDS-PAGE
  • (B) Mixture of macromolecules undergoes electrophoresis through a porous gel.

Staining Proteins in Gels

  • Chemical stains detect proteins based on differential binding of the stain by the protein molecules and the gel matrix.
  • They are nonspecific in action, detecting proteins without regard to their individual identities.
  • Important characteristics for a useful stain:
    • Low background
    • High sensitivity
    • Large linear range
    • Ease of use

Molecular Weight Estimation

  • Molecular weight standards are used for estimation by SDS-PAGE.
  • A standard curve is generated by plotting the log of the molecular weight (log Mr) against the relative migration.
  • The migration of an unknown protein is compared to the standard curve to estimate its molecular weight.