BioChem CH. 5 -...

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Ch. 5 Protein Purification and Characterization Techniques (On 2nd exam) I. Protein Purification A. Breaking Cells 1. Homogenization- Putting cells in a buffer that is hypotonic and squeezing them against a tube wall so they will break and burst 2. Sonication- 3. Freeze-thaw cycles B. Separating Cell Components 1. Differential Centrifugation 2. Pellet and supernatant C. Separating Proteins Based on Solubility 1. "Salting Out" 2. Ammonium Sulfate (removes H20) 3. Precipitation of proteins at different concentrations of (NH4)2 SO4 40% vs 60% 4. Collect precipitates by centrifugation II. Column Chromotography A. Chromatography 1. Stationary Phase (Paper, substance packed in a column) 2. Mobile Phase (Solvent, Elution Buffer) B. Types of Column Chromatography
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1. Gel-filtration chromatography a. separation according to size b. Sephdex (stationary phase) c. Large Molecules elute first d. Elution Profile- graph 2. Affinity Chromatography a. Separation based on affinity for a particular molecule (ligand)
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BioChem CH. 5 -...

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