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choppedspleenMécanique

22 févr. 2014 (il y a 3 années et 4 mois)

90 vue(s)

Metodologías corrientemente utilizadas en
aislamiento y purificación de componentes de
celulares

Basados en el tamaño/

Forma

Centrifugación


Cromatografía de exclusión

PAGE
-
SDS

Basados en la carga/

polaridad

Cromatografía de intercambio iónico

Cromatoenfoque

Cromatografía de afinidad

Solubilidad (sales, pH)

Cromatografía de interac. hidrofóbica

Metodologías específicas

Metodologías generales

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 moti
on
of the centrifuge (centripetal acceleration) makes their path circular. Sedimentation is the process of depositing material t
hat

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
siz
e,
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 t
o
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.


Centrifugation can separate molecules that differ in
mass or density

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


Centrifugation can separate molecules that differ in
mass or density

Electrophoresis separates molecules
according to their charge:mass ratio

SDS
-
polyacrylamide

gel electrophoresis

Two
-
dimensional electrophoresis separates
molecules according to their charge and their
mass

Figure 3
-
42a

Separation of proteins by size: gel filtration
chromatography

Figure 3
-
43a

Separation of components by charge: ion
exchange chromatography

Figure 3
-
43b

Separation of proteins by specific binding
to another molecule: affinity
chromatography

Figure 3
-
43c