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.