LS4Protein Analysis

LS4Protein Analysis - Lecture 4:Protein analysis-how do we...

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Lecture 4:Protein analysis -how do we study proteins? I. Separation How to separate different cellular components? and different proteins? (centrifugation, chromatography, SDS-PAGE, etc) II. Detection (immunological techniques) How to detect a single protein from a mixture of proteins (ELISA,Immunoblot, Immunoprecipitation, etc) III. Sequence and Structural analysis Mass spectrometry, X-ray crystallography, etc Protein Purification A typical mammalian cell contains 3000-5000 different proteins. Some proteins are present at only a few molecules per cell. Other are present at 10 5 or more molecules per cell. To study the structure and properties of a protein of interest, it often must be isolated in a pure form.
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I. Protein separation/purification (2) Centrifugations Differential centrifugation Rate-zonal centrifugation (1) break-up of cells (3) Chromatography ion exchange,gel filtration affinity column, etc The first step in the study of a protein is to separate it from other cellular components, or to isolate it in a pure form. (4) Gel Electrophoresis SDS-PAGE, 2-D gel Physical disruption, Detergent break-up tissues ultrasound French press detergent homogenize
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Differential Differential Centrifugation Centrifugation Particles sediment according to mass and density Supernatant Decant liquid Pellet Particles (or molecules) that sediment rapidly are separated from slower sedimenting materials that remain in the supernatant. The rapidly sedimenting material is collected in the pellet. large particle small particle centrifugal force Separates a mixture of particles (macromolecules, cell organelles) that differ greatly in size. Sample is layered on top of gradient Large particle Smaller particle Sucrose gradient Centrifuge g Stop Centrifuge Puncture hole Collect fractions and assay each. Increasing mass of particles ± A sucrose gradient A sucrose gradient (eg. 10 (eg. 10 -30%) is used to 30%) is used to provide density stability during centrifugation.
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This note was uploaded on 01/16/2010 for the course LIFESCI Life scien taught by Professor Lorenz,t.c. during the Fall '09 term at UCLA.

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LS4Protein Analysis - Lecture 4:Protein analysis-how do we...

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