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09-26 - Lecture 11 Instrumentation Raphael Bar-Or...

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Lecture 11 - Instrumentation Raphael Bar-Or Instrumentation You have now gotten a small glimpse into the complexity of biology How were all of these data collected? New trend in biology – “High Throughput” technology We are in the midst of a revolution! Explosion in data Outline Getting “pure” samples Characterization of phenotypes Characterization of molecules Understanding molecular function Molecular localization
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Science of Biology Controlled studies Qualitative / quantitative measurements { Issues of limits of detection / dynamic range { Calibration { Sensitivity / specificity Laboratory techniques { In-vitro { Ex-vivo { In-vivo { In-silico Getting “Pure” Samples Why study pure samples? { Reproducible { Remove interferences { Simpler / smaller experiments Analogy extends to whole organisms when we get pure strains by inbreeding Usually this is a problem of separation Separations Separating cells and organelles { Centrifuges Can achieve up to 40,000 g’s ! Separate mixtures based on density Cells will form a “pellet”
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Flow Cytometry Used to separate cells based on both physical and receptor characteristics Can be used as a preparative or analytic technique ~ 50-300 μM Flow Cytometry As a preparative method a tiny mechanical switch is added after laser detection to divert cells As an analytic technique it is used to count cells of various types { Fluorescent stain for various proteins Molecular separation Chromatography – Separation of compounds by their chemical properties including { Molecular size (size exclusion) { Polarity (reverse phase) { Ionic tendencies (ion-exchange) { Specific interactions (affinity) { Chirality! (difficult)
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Chromatography Most modern chromatography is done with High Performance Liquid Chromatography (HPLC) Uses the differential attraction of an analyte to a immobilized material in a column. Columns are usually packed with some matrix with huge surface area and many functional groups bonded to the surface.
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