Post on 12-Jan-2016
description
Tissue homogenization
Cell fractionation
Centrifugation
Ruchaneekorn W. Kalpravidh
Research and Development
Upstream Processes
Production
Downstream Processes
Product
Gene Discovery
Cloning and Transformation
Cell Line Development
Media Preparation
Microbial Fermentation
Mammalian Cell Culture
Harvest Cells
Cell Disruption
Protein Purification
Analytical Tests
Harvest Cells
Cell
Disruption
Total Proteins
Pure Protein
Analytical
Tests
Growth Medium
Cell Debris
Unwanted Proteins
Centrifugation
Filtration
Enzymatic
Chemical
Physical
Purification Steps
Methods in cell research
Oldest tool microscope
Microsurgery - means of analyzing cell functions
e.g. the transplantation of a nucleus from
one cell to another, as in amoeba.
relies heavily on techniques of biochemical
analysis to determine the molecular nature
of cell functions and structure.
Modern cell research
Biochemical analysis
- requires large amounts of a purified cell component
depends on the ability of the cell biologists to
lyse cells and fractionate them into their structural
parts.
Cell fractionation methods
involve the homogenization or destruction
of cell boundaries by different mechanical or
chemical procedures, followed by the separation
of the subcellular fractions according to mass,
surface, and specific gravity
Cell Fractionation
the breaking open of cells and separation
of the parts into pure fractions
requires a large number of cells
The breaking open of cells lysis
homogenization
Cell Disruption
Chemical: alkali, organic solvents,
detergents
Enzymatic: lysozyme, chitinase
Physical: osmotic shock, freeze/thaw
Mechanical: sonication, homogenization,
French press
Chemical Disruption
Detergents such as Trition X-100 or NP40 can permeabilize cells by solubilizing membranes.
Detergents can be expensive, denature proteins, and must be removed after disruption
French Press
Cells are placed in a
stainless steel
container. A tight
fitting piston is
inserted and high
pressures are
applied to force cells
through a small hole.
Sonication
A sonicator can be
immersed directly into
a cell suspension.
The sonicator is
vibrated and high
frequency sound
waves disrupt cells.
Homogenization
Cells are placed in a
closed vessel (usually
glass). A tight fitting
plunger is inserted and
rotated with a downward
force. Cells are
disrupted as they pass
between the plunger and
vessel wall.
Homogenization
mechanical disruption of cell membrane
with a homogenizer
cell membrane is sometimes dissolved with
a detergent solution (triton X - 100)
Homogenizer
a tube and a close fitting pestle
The cells are placed in the tube in an
appropriate solutions of inorganic ions and
low MW organic molecules e.g. sucrose.
(disrupt the cells and release the contents
without damaging subcellular organelles)
in order to maintain the functional and structural
properties of the cell parts (once the cells are broken
open)
the pestle is inserted in to the tube and rotated
as it is drawn in and out of the tube
The motion creates a shearing action that
breaks open the cell.
Disrupted Cells
Supernatant
Pellet(discard) Centrifuge
Cell Lysate
Cell-Free LysateProteins, Nucleic Acids,Small Molecules
MultiplePurification Steps
UnwantedMolecules
Pure Protein
Mitochondria, chloroplasts, lysosomes, nuclei,
microbodies, and ribosomes largely intact
GA, cell membrane fragmented
ER small fragments form small
sealed vesicles
microsomes
major structural components
centrifugation
pure fractions
Separation is possible the velocity with which
a particular cell structure sediments in a centrifugal
field depends on size, density, and shape.
Centrifugation
Centrifuge the most versatile tools of
molecular biology
to characterize substances
to separate them
Centrifugation
When a centrifugal force is applied to an
aqueous mixture, components of larger size
and density will sediment faster
Low speed centrifugation is used to separate
intact cells from medium
High speed centrifugation can be used to
separate subcellular components
Fixed-Angle Centrifugation
Swinging-Arm Centrifugation
Differential Centrifugation
supernatant
Analytical ultracentrifuge
information concerning the mass and
(in a limited way) the shape of a molecule
Preparative centrifuge
permit one to use those parameters to
separate molecular types.
Ultracentrifuge
attain higher rotor velocities
contain an optical system, allowing to
observe changes in the solute distribution
occurring in the sample.
Rotors of all UC spin in a vacuum
to prevent heating from air friction
Modern UC velocities up to 70,000 rpm
Centrifugal fields
The force that any particle experiences ina spinning rotor
F = m*w2r
m* = buoyant mass of the particle (i.e., itsmass less than the mass of solvent it displaces)
w = the velocity of the rotor in radians/sec
r = the distance to the particle from thecenter of the rotor.
w2r = radial acceleration or centrifugalacceleration
at 70,000 rpm, a particle 7 cm from the center
a = (70000 rpm x 2p rad/rev x 1/60 min/s)2 (7cm)
= (7329)2 s-2 x 7cm
= 3.76 x 108 cm/s2
normal acceleration of the earth’s gravity (g) = 980 cm/s2
a = 3.76 x 108
980x g
= 384,000 x g
Sedimentation Velocity
Any molecule or particle that is not isodense
with the fluid it displaces will tend to float or sink,
depending on whether it is lighter or heavier than
the surrounding fluid.
The velocity, v, at which a particular substance
moves toward the top or bottom of a liquid column
the acceleration.
The constant of proportionality
= sedimentation coefficient, S:
v = sw2r
S = velocity/unit acceleration
Sedimentation coefficient
(S)
= 2.6 x 10-4 cm/s3.8 x 108 cm/s2
= 7 x 10-13 s
e.g. g-globulin - has a component that
sediments @velocity of 2.6 x 10-4 cm/s
( 0.95 cm/h) @ centrifugal field 384,000 x g
unit 10-13s Svedberg
(Swedish scientist developed UC in 1920s)
7 Svedbergs = 7S
S its buoyant mass and is fastest for spherical
particles
This coefficient as S, is related to MW of the
particle e.g. tRNA 4S, MW 25,000 daltons
Differential centrifugation
cell homogenate (lysate)
500 - 1000 x g 10-15′
pellet - nuclei
10,000-12,000 x g 10-15′
pellet - mitochondria and lysosome
105,000 x g 60′
pellet - ribosomes and ER (microsomes)
supernatant - soluble fraction
Improvement of differential centrifugation
density gradient
discontinuous continuous
layers of varying densities
e.g. Sucrose 1.6 0.5M
mixing of 2 concn
of sucrose
bottom top
gradients is formed
material is layered on top
particles reach equilibrium with the gradient
isopyknic (equal density) centrifugation
To avoid drastic changes in osmotic pressure,
macromolecular media e.g. Ficoll, Percoll
are used.
Improvements in this type of fractionation
the use of heavy water, CsCl and media with
different partition coefficient
use: Zonal rotors form density gradient
while the rotor is spinning
sample is layered and centrifugeduntil the isopyknic zonal layerof the particles is reached.
Buoyant density of macromolecule
i.e. the density at which it will reach an equilibrium with
the suspending medium.
e.g. DNA 2 sources diff buoyant density
band at diff spots in CsCl gradient.