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LE 6-6

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LE 6-6

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  1. Pili Nucleoid Ribosomes LE 6-6 Plasma membrane Cellwall Bacterial chromosome Capsule 0.5 µm Flagella A typical rod-shaped bacterium A thin section through the bacterium Bacillus coagulans (TEM)

  2. ENDOPLASMIC RETICULUM (ER Nuclear envelope Flagellum Rough ER Smooth ER NUCLEUS Nucleolus Chromatin Centrosome Plasma membrane LE 6-9a CYTOSKELETON Microfilaments Intermediate filaments Microtubules Ribosomes: Microvilli Golgi apparatus Peroxisome Mitochondrion In animal cells but not plant cells: Lysosomes Centrioles Flagella (in some plant sperm) Lysosome

  3. Nuclear envelope Rough endoplasmic reticulum NUCLEUS Nucleolus Chromatin Smooth endoplasmic reticulum Centrosome Ribosomes (small brown dots) LE 6-9b Central vacuole Golgi apparatus Microfilaments Intermediate filaments CYTOSKELETON Microtubules Mitochondrion Peroxisome Chloroplast Plasma membrane Cell wall Plasmodesmata Wall of adjacent cell In plant cells but not animal cells: Chloroplasts Central vacuole and tonoplast Cell wall Plasmodesmata

  4. LE 6-5a Homogenization Tissue cells Homogenate Differential centrifugation

  5. Centrifugation allows for Separation

  6. Isolating Organelles by Cell Fractionation • Cell fractionation with centrifugation takes cells apart and separates the major organelles from one another, which enables scientists to determine the functions of organelles

  7. Centrifugation • simple, one-step: separates pellet and supernantant • Differential Centrifugation: multi-step, different speeds, often used for cell fractionation into organelles • Gradient Centrifugation: creates several fractions between density layers, such as sucrose gradient (mitochondria and chloroplasts separation)

  8. Balance Centrifuge Tubes • Tube mass must be evenly distributed for the rotor to spin safely. • Use a tube with matched mass of water to balance a single tube.

  9. rcf and rpm Relative centrifugal force is also called rcf or “x g”. It is a measure of the force exerted on the sample. Revolutions per minute (rpm) is a measure of rotor speed.

  10. rpm and rcf (xg) are different units Experimental centrifugation values are usually expressed in xg rather than rpm. This is because different centrifuges have different size rotors. . A wider rotor will exert higher force on the sample compared to a rotor with a smaller radius. • The relationship between RPM and RCF is as follows: • g = (1.118 × 10-5) R (cm) S2 (rpm) • Where • g is the relative centrifugal force, • R is the radius of the rotor in centimeters, and • S is the speed of the centrifuge in revolutions per minute.

  11. 1000 g (1000 times the force of gravity) 10 min Supernatant poured into next tube 20,000 g 20 min LE 6-5b 80,000 g 60 min Pellet rich in nuclei and cellular debris 150,000 g 3 hr Pellet rich in mitochondria (and chloro- plasts if cells are from a plant) Pellet rich in “microsomes” (pieces of plasma membranes and cells’ internal membranes) Pellet rich in ribosomes

  12. Sucrose Gradient Centrifugation