Biology 2382B Lecture Notes - Lecture 3: Centripetal Force, Cell Membrane, Fluorophore
6 Feb 2017
School
Department
Course
Professor
Document Summary
Lecture 3 - isolation and analysis of cell organelles and molecules. Labeling live cells with fluorescent antibodies or stains. In live cells, antibodies cannot get inside but can bind on anything on the outside (target transmembrane or cell surface proteins) These antibodies are conjugated to a fluorophore. Hoechst is cell permeable and fluorescence can get inside the cell. More of one fluorescence means that more of that particular protein is found on the. Step one: disruption of cell plasma membrane: mechanical homogenization, sonication (ultrasounds, pressure (cells forced through a very narrow valve, non-ionic detergents, placing cells in hypotonic solution (cell will swell and burst) Step two: centrifugation of cell homogenate: differential. A little faster to pull out mt, chloroplasts, lysosomes, and peroxisomes. Faster to pull out plasma membrane, and large polyribosomes. Larger organelles will pallett at lower centripetal force while smaller ones need a faster centripetal force: equilibrium density-gradient.
