Gel Electrophoresis in Protein Isolation

Protein Purification

• Proteins are isolated from various sources, including whole organisms, organs or tissues, embryos, tissue culture cells, and proteins from expression systems.
• Protein purification is necessary to identify the structure and function of the protein of interest, study protein regulation and protein-protein interactions, produce antibodies, and perform structural analysis by X-ray crystallography.

Steps of Protein Purification

• Step 1: Solubilization of protein
  • A. Homogenization:
    • On a laboratory scale: sonication, French Press, enzymatic or chemical cleavage of cells, and freeze-thawing
    • For larger scale: high-pressure homogenization and bead milling
  • B. Centrifugation:
    • Isolation of proteins that are components of the membrane or subcellular assembly
    • Centrifugation speed is adjusted to remove only the cell components denser than the desired organelle
  • C. Filtration
• Step 2: Stabilization of proteins
  • pH: Proteins are placed in buffered solutions at or near their native pH to prevent denaturation or loss of function
  • Temperature: Protein purification is normally carried out at low temperature (~ 0°C), while some proteins are thermally stable at high temperatures
  • Inhibition of proteases
  • Retardation of microbes that can destroy proteins (e.g., using sodium azide)
• Step 3: Isolation of proteins
  • Characteristic-based isolation methods:
    • Solubility: salting out, salting in
    • Molecular size: dialysis, gel filtration chromatography
    • Ionic charge: ion-exchange chromatography, gel electrophoresis, isoelectric focusing
    • Binding specificity: affinity chromatography

Ion-Exchange Chromatography

• Stationary phase: ion-exchangers (e.g., carboxymethyl (CM) group, diethylaminoethyl (DEAE) group)
• Mobile phase: buffers of variable concentrations
• Principle: Attraction between oppositely charged ion-exchangers and analyte (protein sample)
• Steps:
  1. Equilibration: Stabilization of the ion-exchangers with oppositely charged ions in the buffer
  2. Sample application and wash: Protein bound to the ion-exchangers remains attached while other proteins are removed during wash
  3. Elution: Removal of bound protein from the ion exchangers with the help of increased concentration of elution buffer
  4. Regeneration: Preparing the ion exchangers for the next round of protein purification

Gel Filtration Chromatography

• Also known as molecular exclusion chromatography
• Stationary phase: Porous beads (e.g., Sephadex, Sepharose)
• Mobile phase: Buffer (e.g., tetrahydrofuran, chloroform, dimethylformamide)

Gel Electrophoresis

• Gel Electrophoresis Apparatus:
  1. Buffer tank
  2. Buffer
  3. Electrodes
  4. Power supply
  5. Support media
  6. Tracking dye
• Types of Gel Electrophoresis:
  • Horizontal
  • Vertical
• Polyacrylamide gel electrophoresis (PAGE) is the most widely used method in the analysis of complex mixtures of proteins

Polyacrylamide Gel Electrophoresis (PAGE)

• Continuous Gel Electrophoresis:
  • pH is uniform throughout the gel
  • Pore size is uniform throughout the gel
• Discontinuous Gel Electrophoresis:
  • pH differs from one gel to another
  • Pore size varies from one gel to another
• Types of PAGE:
  • Native PAGE: Proteins are run in their native state (i.e., tertiary or quaternary structure)
  • SDS-PAGE: Proteins are denatured with sodium dodecyl sulphate (SDS) and β-mercaptoethanol, which reduces disulphide bridges that stabilize protein tertiary structure

Isoelectric Focusing

• Principle: At the isoelectric point, the net charge of the protein becomes zero, and its mobility in an electric field becomes zero
• Procedure:
  • A cell extract is fully denatured by high concentration of urea (8M)
  • Denatured cell extract is poured on the polyacrylamide gel layered with ampholytes