Cell Lysis Centrifugation

2/24/2010
Cell Lysis, Centrifugation Lysis,
JA Negrn, Ph.D. IAUPR Barranquitas BIOT 4928
Summary
Cell lysis Basic principles of centrifugation Types of centrifugation separation Centrifugation in protein purification
JA Negrn, Ph.D. BIOT 4928
2/24/2010
Cell Lysis
Cell lysis is the first step in cell fractionation and protein purification.
http://www.youtube.com/watch_popup?v=E83XhBGy3nY
Cell lysis or cellular disruption is a cell biology method for the release of biological molecules including organelles, proteins, DNA, RNA and lipids from inside a cell. Many techniques are available for the disruption of cells, including physical and detergent-based methods.
Cell lysis is vital for the extraction of DNA, RNA and Proteins from cells
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 agitation Pressure Sonication Nitrogen bomb or nitrogen burst lysis method Ultrasound with small probes
Mechanical or physical lysis methods include:

Other methods
Chemicals, such as mild detergents Enzymatic hydrolysis
2/24/2010
Traditional Methods of Cell Lysis
Mechanical Lysis
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.
2/24/2010
Liquid Homogenization
Resuspended cells are crushed in a suitable buffer Cells are lysed by forcing the cell or tissue suspension through a narrow space, thereby shearing the cell membranes. Types
Dounce homogenizer Potter-Elvehjem homogenizer French press
Sonicator
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 is applied in multiple short bursts to a sample immersed in an ice bath.
2/24/2010
Freeze/Thaw
Commonly used to lyse bacterial and mammalian cells. The technique involves freezing a cell suspension in a dry ice/ethanol bath or freezer and then thawing the material at room temperature or 37C. This method of lysis causes cells to swell and ultimately break as ice crystals form during the freezing process and then contract during thawing.
Mortar and Pestle

Manual grinding is the most common method used to disrupt plant cells. Tissue is frozen in liquid nitrogen and then crushed using a mortar and pestle. Because of the tensile strength of the cellulose and other polysaccharides comprising the cell wall, this method is the fastest and most efficient way to access plant proteins and DNA.
2/24/2010
Centrifugation: Basic Principles

A centrifuge is a device for separating particles from a solution according to their size, shape density, size shape, density viscosity of the medium and rotor speed.
Centrifugation
Centrifugation produces a centripetal force that can be many hundreds or thousands of times the force of gravity, thus speeding up the process considerably. The greater the number of revolutions per minute (RPM), the greater the force of gravity. The theoretical basis of this technique is the effect of gravity on particles (including macromolecules) in suspension. Two particles of different masses will settle in a tube at different rates in response to gravity. Centrifugal force (measured as xg, gravity) is used to increase this settling rate in an instrument called a centrifuge.
Centrifugal force RPM
2/24/2010
Physics of Centrifugation
Centrifugal force Centripetal force
F = ma F = mw2r
The F is linearly proportional to the mass of the object, but the force increases with the square of the rotation speed.
Motion of body of mass (m) attached m to a string with constrained motion in a circle of radius (r) r
angular velocity Centrifugal force is a consequence of rotation, which implies an acceleration.

Relative centrifugal force (RCF)

Since the motion is circular the acceleration can be calculated as the product of the radius (r) and the square of the angular velocity (2N). Therefore the the acceleration applied to a sample within a centrifuge is called Relative Centrifugal Force (RCF):
RCF = r (2N)2/g
Since g=980 cm/s-2, plus the rotational speed, N, is expressed in rpm, then RCF:
RCF = (1.118 x 10-5)(r in cm)(rpm)2

2/24/2010
g and rpm
The centrifugal force (expressed as # gravities or, # xg) generated is proportional to the rotation rate of the rotor (in rpm) and the distance between the rotor center and the centrifuge tube. A given centrifuge may use multiple rotor sizes to give flexibility in choosing centrifugation conditions.
Nomograph
Each centrifuge has a special graph, a nomograph, or a table which relates rotation rate (rpm) to centrifugal force (xg) for each size of rotor it accepts.
2/24/2010
Beckman J2-HS Centrifuge and Rotor JA-18 J2JARCF (xg) 37 148 333 591 924 1 330 1 810 2 370 2 730 2 990 3 700 4 470 5 320 6 250 7 240 8 320 rpm 500 1 000 1 500 2 000 2 500 3 000 3 500 4 000 4 300 4 500 5 000 5 500 6 000 6 500 7 000 7 500
9 460 000 JA Negrn, Ph.D. BIOT84928
Rotor Speed Calculations
http://www.sciencegateway.org/tools/rotor.htm
2/24/2010
Centrifugation and Speed

Low speed : Below 10,000 rpm High speed : 20,000 to 25,000 rpm Ultrahigh speed : over 25,000 rpm
Classes of centrifuges and their applications
From Cole-Parmer Technical Library

10
2/24/2010
Cellular Markers
Subcellular Fraction Nuclei Mitochondria Lysosomes Microsomes Cytosol Relative Density 1 2 3 4 5 Marker Cromatin Succinate Dehydrogenase Acid Phosphatase Glucose-6Phosphatase Lactate Dehydrogenase
Types of Centrifugation
Differential centrifugation Rate zonal (size) separation
Sucrose gradients Ficoll gradients
Isopycnic separation
CsCl for nucleic acids
11
2/24/2010
Homework
Differences in Analytical Ultracentrifugation vs Preparative Centrifugation
12

Cell Lysis Centrifugation

Încărcat de

Informații document

Descriere originală:

Drepturi de autor

Formate disponibile

Partajați acest document

Partajați sau inserați document

Opțiuni de partajare

Vi se pare util acest document?

Este necorespunzător acest conținut?

Drepturi de autor:

Formate disponibile

Cell Lysis Centrifugation

Încărcat de

Drepturi de autor:

Formate disponibile

2/24/2010

Cell Lysis, Centrifugation Lysis,

JA Negrn, Ph.D. IAUPR Barranquitas BIOT 4928

JA Negrn, Ph.D. BIOT 4928

Mechanical or physical lysis methods include:

Traditional Methods of Cell Lysis

JA Negrn, Ph.D. BIOT 4928

JA Negrn, Ph.D. BIOT 4928

JA Negrn, Ph.D. BIOT 4928

Mortar and Pestle

JA Negrn, Ph.D. BIOT 4928

Centrifugation: Basic Principles

JA Negrn, Ph.D. BIOT 4928

angular velocity Centrifugal force is a consequence of rotation, which implies an acceleration.

Relative centrifugal force (RCF)

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

JA Negrn, Ph.D. BIOT 4928

JA Negrn, Ph.D. BIOT 4928

9 460 000 JA Negrn, Ph.D. BIOT84928

Rotor Speed Calculations

JA Negrn, Ph.D. BIOT 4928

Centrifugation and Speed

JA Negrn, Ph.D. BIOT 4928

Classes of centrifuges and their applications

From Cole-Parmer Technical Library

JA Negrn, Ph.D. BIOT 4928

JA Negrn, Ph.D. BIOT 4928

JA Negrn, Ph.D. BIOT 4928

S-ar putea să vă placă și