Medical Biochemistry for Physiotherapy 2024-2025 PDF

Medical Biochemistry for Physiotherapy 2024-2025 Methods of Protein Separation PROTEINS ARE DIFFERENT 1. Ultracentrifugation Separate 2. Electrophoresis and study 3. Chromatography 1. Ultracentrifugation  Definition: Centrifugation...

Medical Biochemistry for Physiotherapy 2024-2025 Methods of Protein Separation PROTEINS ARE DIFFERENT 1. Ultracentrifugation Separate 2. Electrophoresis and study 3. Chromatography 1. Ultracentrifugation  Definition: Centrifugation is a mechanical process involving the use of the centrifugal force to separate particles from a solution. “Ultracentrifugation) is super fast centrifugation that made the purification of macromolecules possible.  Principle Particles in suspension having different masses will settle to the bottom of a tube at different rates. The remaining liquid that lies above is referred to as THE SUPERNATANT. Heavier components travel “Quickly & Fully” to the bottom of the tube and are called THE PRECIPITATE  Depends on: Centrifugal force causes heavier molecules to settle, or sediment, more quickly than lighter molecules. 2. Electrophoresis  Definition: Separation method based on differential of migration of charged particles in an applied electric field. electro- phoresis means means "electricity” "migration  Principle: Sample is Electric field is deposited on a applied (e.g., gel, cellulose solid support acetate membrane) which is then soaked with a conducting solution(Buffer) The molecules in samples move through the solid support DEPENDS ON: Charge  APPLICATIONS: PLASMA PROTEINS ELECTROPHORESIS POLYACRYLAMIDE GEL ELECTROPHORESIS ISOELECTRIC FOCUSING Plasma proteins electrophoresis Sample is applied to Cellulose Acetate solid support Electrophoresis of sample in electrolyte buffer Plasma proteins electrophoresis Separated bands stained and visualized Densitometer scanning converts bands to peaks The normal pattern of plasma proteins by electrophoresis is …. Characteristic changes in the amounts of one or more of these five SMALLEST bands are found in many diseases. MOST ABUNDANT  Normal pattern Observe the change! Chronic infections Gammaglobulins are increased  Normal pattern Observe the change! Immune deficiency e.g., AIDS (Increased susceptibility to infections) Gammaglobulins are decreased  Normal pattern Observe the change! Liver Cirrhosis Liver function is sufficiently decreased Albumin alpha and beta globulins are decreased Polyacrylamide Gel Electrophoresis (PAGE) Gel is cast vertically between a pair of glass plates Samples are added and the electric current applied Proteins migrate through the pores in the gel. Smaller proteins Larger proteins migrate FASTER migrate SLOWER Isoelectric focusing (IEF) PAGE is set at different Ph values generating a pH gradient. Positively and negatively charged proteins move to the negative and positive electrodes, respectively, until they reach isoelectric points (pI) What is meant by isoelectric point (pI)? The pH at which a molecule carries no net electrical charge or is electrically neutral 3. Chromatography  Definition: A laboratory technique for separation of a sample dissolved in a mobile phase (gas or solvent) which carries it through a fixed system called the stationary phase.  Principle: Different mixture constituents have different affinities for the stationary phase (according to its nature)  Principle: Depending on their interactions with the surface of stationary phase. They will travel at different velocities in the mobile phase, causing them to separate. DEPEND ON: Charge THAT’S ALL!