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2/24/2010

Cell Lysis, Centrifugation Lysis,

JA Negrón, Ph.D. IAUPR Barranquitas BIOT 4928

Summary
• • • • Cell lysis Basic principles of centrifugation Types of centrifugation separation Centrifugation in protein purification

JA Negrón, Ph.D. BIOT 4928

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D. JA Negrón. BIOT 4928 2 . Ph.youtube.com/watch_popup?v=E83XhBGy3nY • Cell lysis or cellular disruption is a cell biology method for the release of biological molecules including organelles. Ph. proteins. such as mild detergents – Enzymatic hydrolysis JA Negrón. RNA and lipids from inside a cell. DNA. • Many techniques are available for the disruption of cells.D. 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. RNA and Proteins from cells • • For most cells. BIOT 4928 Cell lysis is vital for the extraction of DNA.2/24/2010 Cell Lysis • Cell lysis is the first step in cell fractionation and protein purification. – http://www. including physical and detergent-based methods.

D. BIOT 4928 3 . Ph.2/24/2010 Traditional Methods of Cell Lysis JA Negrón. BIOT 4928 Mechanical Lysis • Cell envelope is physically broken. JA Negrón.D. Ph. 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.

Ph.D. BIOT 4928 4 . BIOT 4928 Sonicator • Cells are disrupted by energy produced by sound waves • Pulsed. ultrasonic treatment is applied in multiple short bursts to a sample immersed in an ice bath. JA Negrón. thereby shearing the cell membranes. Ph. • Types – Dounce homogenizer – Potter-Elvehjem homogenizer – French press JA Negrón.D. high frequency sound waves to agitate and lyse cells • To prevent excessive heating.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.

BIOT 4928 5 . • This method of lysis causes cells to swell and ultimately break as ice crystals form during the freezing process and then contract during thawing. JA Negrón. • Because of the tensile strength of the cellulose and other polysaccharides comprising the cell wall.D. BIOT 4928 Mortar and Pestle • Manual grinding is the most common method used to disrupt plant cells. Ph. • The technique involves freezing a cell suspension in a dry ice/ethanol bath or freezer and then thawing the material at room temperature or 37°C. • Tissue is frozen in liquid nitrogen and then crushed using a mortar and pestle.D.2/24/2010 Freeze/Thaw • Commonly used to lyse bacterial and mammalian cells. this method is the fastest and most efficient way to access plant proteins and DNA. JA Negrón. Ph.

size shape. • Centrifugal force (measured as xg. BIOT 4928 Centrifugation • Centrifugation produces a centripetal force that can be many hundreds or thousands of times the force of gravity. shape density. density viscosity of the medium and rotor speed.2/24/2010 Centrifugation: Basic Principles • A centrifuge is a device for separating particles from a solution according to their size. Ph. thus speeding up the process considerably.D. Two particles of different masses will settle in a tube at different rates in response to gravity. • The theoretical basis of this technique is the effect of gravity on particles (including macromolecules) in suspension. Centrifugal force α RPM JA Negrón. JA Negrón. BIOT 4928 6 . gravity) is used to increase this settling rate in an instrument called a centrifuge. The greater the number of revolutions per minute (RPM). the greater the force of gravity.D. Ph.

D. 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.118 x 10-5)(r in cm)(rpm)2 JA Negrón. Ph. N. which implies an acceleration.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.D. Therefore the the acceleration applied to a sample within a centrifuge is called Relative Centrifugal Force (RCF): RCF = r (2πN)2/g • Since g=980 cm/s-2. BIOT 4928 7 . JA Negrón. plus the rotational speed. is expressed in rpm. then RCF: RCF = (1. BIOT 4928 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 (2πN). Ph.

JA Negrón. Ph.D. BIOT 4928 Nomograph • Each centrifuge has a special graph.2/24/2010 g and rpm • The centrifugal force (expressed as # gravities or. BIOT 4928 8 . a nomograph. # xg) generated is proportional to the rotation rate of the rotor (in rpm) and the distance between the rotor center and the centrifuge tube.D. or a table which relates rotation rate (rpm) to centrifugal force (xg) for each size of rotor it accepts. JA Negrón. • A given centrifuge may use multiple rotor sizes to give flexibility in choosing centrifugation conditions. Ph.

sciencegateway. BIOT 4928 9 .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 Negrón.org/tools/rotor.D. BIOT84928 Rotor Speed Calculations http://www. Ph.htm JA Negrón. Ph.D.

2/24/2010 Centrifugation and Speed • Low speed : Below 10.000 to 25. BIOT 4928 10 . Ph. BIOT 4928 Classes of centrifuges and their applications From Cole-Parmer Technical Library JA Negrón.D.000 rpm • High speed : 20.D.000 rpm JA Negrón.000 rpm • Ultrahigh speed : over 25. Ph.

D.D. Ph. Ph. BIOT 4928 11 . BIOT 4928 Types of Centrifugation • Differential centrifugation • Rate zonal (size) separation – Sucrose gradients – Ficoll gradients • Isopycnic separation – CsCl for nucleic acids JA Negrón.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 JA Negrón.

D.2/24/2010 Homework • Differences in Analytical Ultracentrifugation vs Preparative Centrifugation JA Negrón. BIOT 4928 12 . Ph.