TR

SDS-Page

Overview

  • SDS-Page: process by which proteins are separated based on their molecular weights
  • Proteins are made of smaller units (monomers) called amino acids.
    • There are 20 common amino acids.
    • The sequence and interaction between these different amino acids determine the function of the protein they form.
    • Amino acids are joined together by peptide bonds to form polypeptide chains.
    • Chains of amino acids constitute a protein.
  • The “R” group may be charged or uncharged, or may be a long side chain.
    • Thus, each amino acid has different properties and can interact with other amino acids in the chain.
  • A molecule’s electrical charge and its mass affect its mobility through a gel during electrophoresis.
  • Charge density: the ratio of charge to mass.
  • Since every protein is made of a unique combination of amino acids, each of which may have a positive, negative, or neutral charge, the net charge of each protein is naturally different.
  • The inherent charges of proteins must be removed as a factor affecting migration in order for polyacrylamide electrophoresis to be effective as a method of protein molecular weight determination.
  • The intrinsic charges of proteins are obscured by placing a strongly anionic (negatively charged) detergent, sodium dodecyl sulfate (SDS), in both the sample buffer and the gel running buffer.
  • SDS binds to and coats the proteins and also keeps them denatured as relatively linear chains.
    • In this form, proteins migrate in a polyacrylamide gel as if they have equivalent negative charge densities, and mass becomes the main variable affecting the migration rate of each protein.
  • To effectively determine the molecular weights of proteins, the secondary, tertiary, and quaternary structures of the protein complexes within a protein extract are disrupted prior to electrophoresis.
  • A polyacrylamide gel is positioned in a buffer-filled chamber between two electrodes, protein samples are placed in wells at the top of the gel, and the electrodes are connected to a power supply that generates a voltage gradient across the gel.
  • The SDS-coated, negatively charged proteins migrate through the gel away from the negatively charged anode toward the cathode, with the larger proteins moving more slowly than the smaller proteins.

Visualizing Proteins

  • Proteins in the samples are not visible while the gel is running.
  • The only visible proteins will be those in the Precision Plus Protein Kaleidoscope standard that have been prestained with covalently attached dyes.
  • If the electric current is left on for too long, the proteins will run off the bottom of the gel.
    • To guard against this and to show you the progress of your gel if you did not have the standards, a blue tracking dye is negatively charged and is also drawn toward the positive electrode.
    • Since the dye molecules are smaller than the proteins expected in most samples, they move ahead of the proteins in the gel.
  • Coomassie Blue stain: binds specifically to proteins and not to other macromolecules such as DNA or lipids.
    • The larger the amount of protein, the more intense the blue staining.