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.