Gel Filtration Chromatography

Stationary Phase and Size Exclusion Chromatography

• Stationary phase composed of cross-linked gel particles with controlled pore size.
• Smaller molecules enter the pores and are delayed in elution time, while larger molecules do not enter and elute from the column before smaller ones.

Ion Exchange Chromatography

• interaction based on overall charge (less specific than affinity).
• Cation exchange: binds to +vely charged groups.
• Anion exchange: binds to –vely charged groups.
• Cationic exchange using a gradient of increasing concentration of NaCl as eluent.

Ion Exchange Chromatography Process

• Proteins are applied to the column, and those with no net charge or a net negative charge pass through.
• Proteins with a net positive charge stick to the column, displacing the Na+ counterions.
• An excess of Na+ ions is added to the column, outcompeting the bound proteins for the binding sites, and the proteins elute.

Enzyme Activity and Specific Activity

• Enzyme activity: moles of substrate converted per unit time = rate × reaction volume.
• Specific activity: enzyme activity per milligram of total protein expressed in μmol min-1mg-1.
• Specific activity increases, while total protein content decreases during purification.

Tracking Proteins during Purification

• Purification is a multi-step process.
• Methods to track proteins include:
  • Enzyme activity assays
  • Western blot or ELISA (for antibody detection)
  • Size determination (not as specific)
  • Mass spectrometry
  • N-terminal sequencing

Protein Extraction from Cells

• Many different proteins exist within one cell.
• Steps needed to extract the protein of interest:
  • Homogenization (physical disruption of cell to release proteins; lysis of cells)
  • Differential centrifugation to recover a clean protein fraction
  • Salting out (e.g., using ammonium sulfate)

Column Chromatography

• Basis of chromatography: different compounds distribute themselves to a varying extent between two phases.
• Proteins in solution interact with the stationary phase (resin in column) and the mobile phase (solvent passing through the column).

Purification using Column Chromatography

• Size-Exclusion/Gel-Filtration chromatography: separates molecules based on size.
• Ion Exchange chromatography: separates molecules based on charge.
• Affinity chromatography: separates molecules based on specific interactions.

Guidelines for Protein Purification

• Define objectives
• Define properties of target protein and critical contaminants
• Minimize the number of steps
• Use a different technique at each step
• Develop analytical assays

Purity of Protein

• Required purity depends on the application:
  • Therapeutic use, in vivo studies: extremely high (> 99%)
  • Biochemical assays, X-ray crystallography: high (95-99%)
  • N-terminal sequencing, antigen for antibody production, NMR: moderately high (< 95%)