1. Metodologías corrientemente utilizadas en aislamiento y purificación de componentes de celulares Basados en el tamaño/ Forma Basados en el tamaño/ Forma Centrifugación Cromatografía de exclusión PAGE-SDS Basados en la carga/ polaridad Basados en la carga/ polaridad Cromatografía de intercambio iónico Cromatoenfoque Cromatografía de afinidad Solubilidad (sales, pH) Cromatografía de interac. hidrofóbica Metodologías específicas Metodologías generales

2. A centrifuge is an instrument that separates particles in solution by spinning them around in a circle. The inertia of the particles helps to separate or sediment them. The particles will tend to move in a straight path due to inertia, but the motion of the centrifuge (centripetal acceleration) makes their path circular. Sedimentation is the process of depositing material that settles to the bottom of a liquid. In centrifugation, the largest and most dense particles will settle to the bottom and the smaller, less dense ones will be in layers above them. A centrifuge generally consists of a compartment that is spun about a central axis, which results in the separation. The separation of the particles is affected their physical properties such as size, shape and density. The viscosity of the solution and the speed at which the centrifuge spins also has an impact on the separation of particles. Some the different physical aspects of centrifugation are: o Angular velocity o Radial acceleration, also called centripetal acceleration o Terminal speed o Resistive Forces Angular velocity, w, is the ratio of angular displacement to the amount of time. A tachometer is an instrument that is used to measure angular velocity of centrifuges. The radial acceleration, or centripetal acceleration is the inward force that is required to keep a particle moving in a circular path. A particle moving in a circular path can be shown to have acceleration toward the center of the circle along a radius. The terminal speed of a particle moving through the solution is reached when the net force of frictional resistance is zero. Centrifugation has several different applications in different fields. In biology it can be used to separate cells and their organelles, viruses, proteins and nucleic acids. It can also be used to separate the various parts of blood. In chemistry centrifuges can be used to find molecular weights of substances and they can separate gases and elements. Centrifugation can be classified in several ways, but the two most prevalent types are analytical and preparative. The properties of sedimenting particles are measured in analytical centrifugation. Some of the properties that can be measured are the sedimentation coefficient and the molecular weight. The sedimentation coefficient is the rate at which the particle sediments in a standard centrifugal field. A general explanation of preparative centrifugation is the isolation of certain particles for reuse.

3. Centrifugation can separate molecules that differ in mass or density

5. Físicos-mecánicos potter, unimixer, french press, sonicación, congelado con N 2 liquido y mortero. Químicos-enzimáticos: tratamiento con proteasas, glucanasas, pectinasas, lisozima. EDTA-detergentes. métodos de lisis celular

6. Centrifugation can separate molecules that differ in mass or density

7. Electrophoresis separates molecules according to their charge:mass ratio SDS-polyacrylamide gel electrophoresis

9. Two-dimensional electrophoresis separates molecules according to their charge and their mass Figure 3-42a

11. Separation of proteins by size: gel filtration chromatography Figure 3-43a

13. Separation of components by charge: ion exchange chromatography Figure 3-43b

15. Separation of proteins by specific binding to another molecule: affinity chromatography Figure 3-43c