Lecture 2 Flashcards
(19 cards)
What is the primary objective of cell disruption?
To obtain intracellular fluid by opening the cell wall, without disrupting any of its components.
What are the two main classifications of tissue and cell disruption methods?
Mechanical and Non-mechanical.
List three categories of non-mechanical cell disruption methods.
Physical (e.g., Temperature, USN), Chemical (e.g., Detergents), and Biological (e.g., Enzymes).
Name three mechanical methods for cell homogenization.
Freezing and crushing (liquid Nitrogen), Microfluidizer (shear pressure), Ultrasonication (high-frequency sound vibration), French press (High Pressure Homogenization, HPH), Rotating blades, Bead-beating/milling, Osmotic Shock, Pestle and Mortar.
How do detergents disrupt cell membranes?
Detergents, such as SDS, solubilize the lipid component of cell membranes.
Give an example of an enzyme used for cell lysis and its function.
Lysozyme hydrolyzes the sugar component of bacterial cell walls. Other examples include lysostaphin, zymolase, cellulose, protease, or glycanase.
How does bead milling achieve cell disruption?
Cell disruption is induced through shear forces produced during the rotary movement of the cells and the beads, cell grinding between beads, and bead-cell collisions.
Explain the principle of ultrasonication for cell disruption.
Ultrasonication creates cavitation by introducing ultrasonic waves, forming vapor bubbles that collapse suddenly and powerfully, damaging the cell wall.
What is centrifugation?
A technique that involves the application of centrifugal force to separate particles from a solution based on their size, shape, density, viscosity of the medium, and rotor speed.
What are the two traditional types of rotors used in centrifuges?
Swinging-bucket and Fixed-angle.
What does RCF stand for, and how does it change within a rotor tube?
RCF stands for Relative Centrifugal Force (g-force). The RCF in a rotor tube increases linearly with the radius.
What is differential centrifugation, and what is its main outcome?
Differential centrifugation separates components based on their size and density, producing enriched fractions of subcellular organelles.
What types of particles sediment first at low speeds and short times in differential centrifugation?
Unbroken cells and nuclei, as they are the largest and heaviest subcellular structures.
How did De Duve identify subcellular fractions?
By using marker enzymes, as each subcellular structure has a unique biochemical composition including component enzymes
Give an example of a marker enzyme for mitochondria.
Cytochrome C oxidase and succinate dehydrogenase.
What are the disadvantages of differential centrifugation?
Poor resolution and recovery due to particle size heterogeneity.
How do density gradient centrifugations improve resolution?
They separate particles based on differences in their density relative to the gradient, often involving ultracentrifuges.
What are the two types of density centrifugation, and what does each primarily rely on for separation?
Rate Zonal Centrifugation: Relies on size and shape (particle density > highest gradient density).
* Isopycnic Banding: Relies on density (gradient density > densest particle density).
What is a critical safety precaution for using a centrifuge?
Always balance the tubes to avoid damaging the centrifuge or yourself.
