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PROTEIN PURIFICATION AND ANALYSIS

PROTEIN PURIFICATION AND ANALYSIS. Assays Need measures for the object (enzyme activity, chromophore, etc.) and for total protein concentration:. Use spectrophotometry for: Measuring substrate or product concentration if one is “colored” (absorbs UV or visible light).

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PROTEIN PURIFICATION AND ANALYSIS

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  1. PROTEIN PURIFICATION AND ANALYSIS

  2. Assays Need measures for the object (enzyme activity, chromophore, etc.) and for total protein concentration:

  3. Use spectrophotometry for: Measuring substrate or product concentration if one is “colored” (absorbs UV or visible light). 2. Measuring substrate or product concentration if one reacts with something to form a colored compound. 3. Measuring the concentration of total protein (by UV absorbance: A280 ~ 1 @ 1 mg/ml) or of a specific protein (like hemoglobin) that contains a colored “chromophore.” Measuring the concentration of protein by reacting it with a reagent to form a colored compound. (E.g., cys, tyr, trp reduce Cu2+ to Cu+, which forms an intense blue with BCA; Coomassie reagent turns blue in hydrophobic environment.)

  4. Measuring purification Specific activity (SA) = total enzyme activity/total protein or activity concentration/protein concentration 1 IU (international unit) = 1 µmol substrate used (product formed)/min-mg protein “Purification” at step x = SA of step x / SA of crude extract One measure of “purity” is constant SA. Note: if the protein of interest is 0.2% of the total, then you require 500-fold purification for purity; if each step gives 3-fold purification, then you need 5-6 steps; if each step causes some loss of the protein of interest (e.g. 50%), you need to start with 30-60-times more enzyme than you will recover.

  5. Purification techniques: size, charge, specific binding Size: exclusion chromatography

  6. Two-dimensional gel electrophoresis: IEF followed by SDS-PAGE

  7. Affinity chromatography: separate by biological specificity • 1. Attach “ligand” to insoluble column material • 2. Combine protein mixture with column: enzyme binds to ligand • 3. Wash column to remove unwanted material • 4. Elute enzyme with substrate or change in pH (to reduce binding)

  8. Summary Enzyme purification involves: breaking cells and stabilizing pH, proteolysis, measuring enzyme activity and protein concentration, a series of separation techniques Spectrophotometry is useful for measuring protein concentration and, often, enzyme activity Exclusion chromatography, electrophoresis, isoelectric focusing, and affinity chromatograph are useful methods for purifying proteins

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