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Seminario de bioquímica Universidad de Córdoba
- 2. INDEX Characteristics & applications Main methods of protein separation ·Electrophoresis and subtypes ·Cromatography and subtypes ·Dialysis ·Ultracentrifugation
- 3. CHARACTERISTICS AND APPLICATIONS Proteins are very diverse biomolecules. They differ in size, shape, charge, hydrophobicity and affinity for other molecules. All this properties can be used to separate one from the others, so they can be studied separately. Applications: Investigation Forensic Medicine Clinical diagnostics
- 5. ELECTROPHORESIS It consists of the migration of protein molecules contained in a solution when an electric field is applied. The speed at which these molecules move depends on their charge, shape and size. Kinds of electroforesis: -moving front electrophoresis -zone electrophoresis
- 6. MOVING FRONT ELECTROPHORESIS The substances to be separated are placed in a U-shaped tube, disolved in an appropriate pH buffer and with an adequate ionic force. Electrodes are placed on the arms of the device, in which an electric field is created. Different proteins move at different speeds depending on their load and friction coefficients. A kind of clouds form and move in the buffer solution. These can be observed with various optical systems. cathode anode
- 7. ZONE ELECTROPHORESIS The sample is required to move on a solid substrate such as paper or certain gels. ● Paper and cellulose acetate electrophoresis ● Gel electrophoresis (agarose, polyacrilamide…) Gel: it is an intermediate state between the solid and the liquid. It has porus of different dimensions that delimit the speed.
- 8. DENATURATING ELECTROPHORESIS IN POLYACRILAMIDE GELS STACKING/ RUNNING GEL ·Acrylamide/Bis ·Tris-HCl ·pH 6.8/8.8 ·SDS ·APS/TEMED The total percentage of acrylamide and the interlacing compound (bisacrylamide) determines the pore size Serves to pass the liquid to gel (Polymerization reaction) Disorganizes the protein by eliminating its secondary and tertiary structures. SDS also envelops the negatively charge proteins.
- 9. LOADING BUFFER ·Tris-HCl ·SDS ·Glycerol ·B-mercaptoetanol ·Bromophenol blue DENATURATING ELECTROPHORESIS IN POLYACRILAMIDE GELS Increases the density of the sample Reduces the disulfure bridg Marks the electrophoresis front
- 10. DENATURATING ELECTROPHORESIS IN POLYACRILAMIDE GELS Proteins with higher molecular weights find it more difficult to move along the gel and lag behind smaller proteins. Marker s Sample s
- 11. CHROMATOGR APHY It is a separation method in which the proteins are distributed in two inmiscible phases: a mobile phase moving in a defined direction and a fixed or stationary phase. Kinds of cromatography depending on where the ststionary phase is located: Planar chromatography (paper, thin layer) Column chromatography (normal phase, affinity, inverse…)
- 13. DIALYSIS A process that separates proteins from solvents by taking advantage of the larger size of proteins. The partially purified extract is placed in a sac. The membrane allows the exchange of salt and buffer, but not of large proteins within the sac.
- 14. ULTRACENTRIFU GATION Ultracentrifugation is an important technique for separating and analyzing proteins of interest based on size and mass. During centrifugation the molecules pass from the top to the bottom of the solution in a tube. At equilibrium, the net effect of gravitational and flotation forces will cause the accumulation of specific proteins at a specific point in the tuve.