Lecture 8 - Lecture 8 Protein purification and analysis 1....

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Lecture 8 Protein purification and analysis 1. Initial steps: size, charge/solubility; Assays: activity and protein; specific activity 2. Affinity purification 3. Cloning (in bacteria, yeast, insect cells) with specific induction and secretion Basic/initial steps 1. Acquire/prepare tissue (1 g = 10 6 plant cell, 10 9 animal cells, 10 12 bacteria) 2. Suspend in buffer (pH stabilizers, solubilizers/detergents, protease inhibitors: PMSF, leupeptin, chymostatin) 3. Lyse cells (Waring blendor, sonicator, French press, mortar and pestle) 4. Centrifuge (e.g. 12 kg x 30 min) lipid supernatant = “crude extract” pellet 5. (NH 4 ) 2 SO 4 or pH precipitations: (“salting out”: see Fig. 5.7) salt competes for water of solvation, precipitating proteins: different proteins at different concentrations (e.g., 20%, 50%, 80% of saturation); 2-3-fold purification Assays Spectrophotometry (for activity, protein concentration, etc.) light source cuvette with photocell Io sample I A = - log 10 (I/I o ) A = a*c*l
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This note was uploaded on 09/29/2010 for the course VEN 101C 91861 taught by Professor Davidsmart during the Spring '09 term at UC Davis.

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Lecture 8 - Lecture 8 Protein purification and analysis 1....

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