cell lysis centrifugation

7/30/2019 Cell Lysis Centrifugation

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CellCell LysisLysis, Centrifugation, Centrifugation

JA Negrn, Ph.D.

IAUPR Barranquitas

BIOT 4928

JA Negrn Ph.D. BIOT 4928

SummarySummary

Cell lysis

Basic principles of centrifugation

Types of centrifugation separation

Centrifugation in protein purification


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Cell lysis is the first step in cell fractionation andprotein purification. http://www.youtube.com/watch_popup?v=E83XhBGy3nY

Cell lysis or cellular disruption is a cell biologymethod for the release of biological moleculesincluding organelles, proteins, DNA, RNA andlipids from inside a cell.

Many techniques are available for the

disruption of cells, including physical anddetergent-based methods.

CellCell LysisLysis


For most cells, mild osmosis is usually enough to lyze the cells.

Important factors

Number of cell samples to lyze

Difficulty of cells to lyze

Efficiency of Cell Lysis

Cell Lysis vs Molecule to be Isolated

Mechanical or physical lysis methods include:

Mechanical agitation

Pressure

Sonication

Nitrogen bomb or nitrogen burst lysis method

Ultrasound with small probes

Other methods

Chemicals, such as mild detergents

Enzymatic hydrolysis

CellCell lysislysis is vital for the extraction ofis vital for the extraction of

DNA, RNA and Proteins from cellsDNA, RNA and Proteins from cells


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Traditional Methods of CellTraditional Methods of Cell LysisLysis


Cell envelope is physically broken,

releasing all intracellular

components into the surrounding

medium

Rotating blades to grind and

disperse large amounts of complex

tissue

Product of interest must be

separated from a complex mixture

Most proteins will be denaturated

by the heat generated unless the

device is sufficiently cooled.

MechanicalMechanical LysisLysis


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Resuspended cells arecrushed in a suitable buffer

Cells are lysed by forcingthe cell or tissue suspensionthrough a narrow space,thereby shearing the cellmembranes.

Types Dounce homogenizer

Potter-Elvehjem homogenizer

French press

Liquid HomogenizationLiquid Homogenization


Cells are disrupted by energy

produced by sound waves

Pulsed, high frequency sound

waves to agitate and lyse

cells

To prevent excessive heating,

ultrasonic treatment isapplied in multiple short

bursts to a sample immersed

in an ice bath.

SonicatorSonicator


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Commonly used to lysebacterial and mammalian cells.

The technique involves freezinga cell suspension in a dryice/ethanol bath or freezer andthen thawing the material atroom temperature or 37C.

This method of lysis causes cellsto swell and ultimately break asice crystals form during the

freezing process and thencontract during thawing.

Freeze/ThawFreeze/Thaw


Manual grinding is the mostcommon method used todisrupt plant cells.

Tissue is frozen in liquidnitrogen and then crushedusing a mortar and pestle.

Because of the tensilestrength of the cellulose andother polysaccharides

comprising the cell wall, thismethod is the fastest andmost efficient way to accessplant proteins and DNA.

Mortar and PestleMortar and Pestle


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A centrifuge is a device for separating

particles from a solution according to their

sizesize, shapeshape, densitydensity, viscosityviscosity of the medium

and rotorrotor speed.speed.

Centrifugation: Basic PrinciplesCentrifugation: Basic Principles


Centrifugation produces a centripetal force that can bemany hundreds or thousands of times the force ofgravity, thus speeding up the process considerably. Thegreater the number of revolutions per minute (RPM),the greater the force of gravity.

The theoretical basis of this technique is the effect ofgravity on particles (including macromolecules) insuspension. Two particles of different masses willsettle in a tube at different rates in response to gravity.

Centrifugal force (measured as xg, gravity) is used toincrease this settling rate in an instrument called acentrifuge.

CentrifugationCentrifugation

Centrifugal forceCentrifugal force RPMRPM


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Physics of CentrifugationPhysics of Centrifugation

F = mw2r

F = ma

The FF is linearly

proportional to

the mass of theobject, but the

force increases

with the square

of the rotation

speed.

Centrifugal

force

Centripetal

force

Motion of body of mass (mm) attached

to a string with constrained motion in

a circle of radius (rr)

angular velocity

Centrifugal force is a consequence of rotation,

which implies an acceleration.


Relative centrifugal force (RCF)Relative centrifugal force (RCF) Since the motion is circular the acceleration can be

calculated as the product of the radius (r) and thesquare of the angular velocity (2N). Therefore the

the acceleration applied to a sample within acentrifuge is called Relative Centrifugal Force(RCF):

RCF = r (2N)2/g

Since g=980 cm/s-2

, plus the rotational speed, N, isexpressed in rpm, then RCF:

RCF = (1.118 x 10-5)(r in cm)(rpm)2


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The centrifugal force (expressed as #gravities or, # xg) generated is proportionalto the rotation rate of the rotor (in rpm) andthe distance between the rotor center andthe centrifuge tube.

A given centrifuge may use multiple rotorsizes to give flexibility in choosingcentrifugation conditions.

g and rpmg and rpm


Each centrifugehas a specialgraph, anomograph, or atable whichrelates rotationrate (rpm) tocentrifugal force

(xg) for each sizeof rotor itaccepts.

NomographNomograph


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BeckmanBeckman J2J2--HSHS CentrifugeCentrifuge and Rotor JAand Rotor JA--1818

RCF (xg)rpm

37 500

148 1 000

333 1 500

591 2 000

924 2 500

1 330 3 000

1 810 3 500

2 370 4 000

2 730 4 300

2 990 4 500

3 700 5 000

4 470 5 500

5 320 6 000

6 250 6 500

7 240 7 000

8 320 7 500

9 460 8 000


Rotor Speed CalculationsRotor Speed Calculations

http://www.sciencegateway.org/tools/rotor.htm


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Low speed : Below 10,000 rpm

High speed : 20,000 to 25,000 rpm

Ultrahigh speed : over 25,000 rpm

Centrifugation and SpeedCentrifugation and Speed


Classes of centrifuges and theirClasses of centrifuges and their

applicationsapplications

From Cole-Parmer Technical Library


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Cellular MarkersCellular Markers

SubcellularSubcellular

FractionFraction

RelativeRelative

DensityDensityMarkerMarker

Nuclei 1 Cromatin

Mitochondria 2Succinate

Dehydrogenase

Lysosomes 3 Acid Phosphatase

Microsomes 4Glucose-6-

Phosphatase

Cytosol 5Lactate

Dehydrogenase


Differential centrifugation

Rate zonal (size) separation

Sucrose gradients

Ficoll gradients

Isopycnic separation

CsCl for nucleic acids

Types of CentrifugationTypes of Centrifugation


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Differences in Analytical

Ultracentrifugation vs Preparative

Centrifugation

HomeworkHomework

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