BAT2

• Electrophoresis


  A technique used in biochemistry, molecular biology and analytical chemistry to separate charged molecules, such as DNA, RNA, proteins and other macromolecules, based on their size and electric charge
• Electrophoresis is based on the principle of the movement / migration of charged particles or molecules in a medium under the influence of an applied electric field
• Agarose gel electrophoresis


  1. Preparation of agarose gel

  2. Loading of samples

  3. Electrophoresis

  4. Visualization
• Agarose gel electrophoresis


  • Rapidly separates molecules into discrete bands based upon charge, size and shape
• Native PAGE (Polyacrylamide gel electrophoresis)

  1. Preparation of the Gel

  2. Sample preparation

  3. Loading the Gel

  4. Electrophoresis

  5. Visualization
• Native PAGE


  Technique used to separate proteins based on their size and charge under native conditions, meaning the proteins retain their native structure and function
• SDS-PAGE (Sodium dodecyl sulfate polyacrylamide gel electrophoresis)

  1. Preparation of Gel

  2. Sample preparation

  3. Loading and electrophoresis

  4. Visualization
• SDS-PAGE


  Widely used technique for separating proteins based on their molecular weights and provides valuable information about protein composition, purity and molecular weight composition
• Capillary electrophoresis


  1. Use of a narrow capillary tube as the separation medium

  2. Introduction of sample into the capillary tube

  3. Application of high voltage across the capillary tube

  4. Separation of analytes based on charge-to-size ratio

  5. Detection of separated analytes
• Capillary electrophoresis


  Used to separate and analyze ions and molecules based on their size and charge
• Isoelectric focusing (IEF)


  1. Preparation of Gel

  2. Sample loading

  3. Applying electric field

  4. Focusing

  5. Separation
• Isoelectric focusing (IEF)


  Technique used to separate proteins or peptides based on their isoelectric points (pI)
• The principle behind the isoelectric focusing is based on migration of charged molecules in pH gradient under the influence of an electric field
• Gel electrophoresis


  1. Preparation of gel

  2. Sample loading

  3. Applying electric field

  4. Focusing

  5. Separation
• Isoelectric point (pI)


  The pH at which a protein carries no net charge
• At the isoelectric point, the protein has no net charge and therefore no longer migrates in the electric field
• 2D gel electrophoresis


  1. Isoelectric focusing (first dimension)

  2. SDS-PAGE (second dimension)
• Affinity chromatography


  • Purification of biomolecules based on specific interactions with an immobilized ligand
  • Ligand acts as 'bait' to selectively capture and retain the target molecule
• Affinity chromatography procedure


  1. Selection of ligand

  2. Immobilization

  3. Addition of sample

  4. Washing

  5. Elution
• Ion-exchange chromatography


  • Separation and purification of charged molecules based on reversible exchange of ions between the sample and the stationary phase
• Ion-exchange chromatography procedure


  1. Stationary phase

  2. Sample addition

  3. Ion exchange

  4. Elution
• Gel-filtration (size-exclusion) chromatography


  • Separation of molecules based on their molecular weight/size
  • Larger molecules pass through the column more quickly, smaller molecules take longer to elute
• Gel-filtration chromatography procedure


  1. Stationary phase

  2. Mobile phase

  3. Sample loading

  4. Separation

  5. Elution