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
Centrifugation
Filtration
Growth Medium
Cell
Disruption
Cell Debris
Enzymatic
Chemical
Physical
Total Proteins
Unwanted Proteins
Purification Steps
Pure Protein
Analytical
Tests
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.
Modern cell research
relies heavily on techniques of biochemical
analysis to determine the molecular nature
of cell functions and structure.
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 Cell Lysate
Pellet
(discard)
Centrifuge
Supernatant Cell-Free Lysate
Proteins, Nucleic Acids,
Small Molecules
Unwanted
Molecules
Multiple
Purification Steps
Pure Protein
Mitochondria, chloroplasts, lysosomes, nuclei,
microbodies, and ribosomes
GA, cell membrane
ER
small fragments
largely intact
fragmented
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 in
a spinning rotor
m*w2r
m* = buoyant mass of the particle (i.e., its
mass less than the mass of solvent it
displaces)
w = the velocity of the rotor in radians/sec
r
= the distance to the particle from the
center of the rotor.
w2r = radial acceleration or centrifugal
acceleration
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 earths gravity (g) = 980 cm/s2
a = 3.76 x 108 x g
980
= 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
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
Sedimentation coefficient = 2.6 x 10-4 cm/s
3.8 x 108 cm/s2
(S)
= 7 x 10-13 s
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
layers of varying densities
e.g. Sucrose 1.6
0.5M
bottom top
continuous
mixing of 2 concn
of sucrose
gradients is formed
material is layered on top
particles reach equilibrium with the gradient
isopyknic (equal density) centrifugation
Improvements in this type of fractionation
the use of heavy water, CsCl and media with
different partition coefficient
To avoid drastic changes in osmotic pressure,
macromolecular media e.g. Ficoll, Percoll
are used.
use: Zonal rotors
form density gradient
while the rotor is spinning
sample is layered and centrifuged
until the isopyknic zonal layer
of 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.
