233_protein_purification_techniques

233_protein_purification_techniques - Chemistry 233...

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Chemistry 233 Fundamentals of Biochemistry Protein Purification Techniques
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Protein Purification Techniques Topics: source of protein making a lysate salting out techniques chromatographic techniques ion exchange gel filtration affinity chromatography electrophoresis techniques SDS electrophoresis isoelectric focusing
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Purification scheme: A series of steps used to purify a protein from a source source of your protein? considerations: concentration of protein in the source ease of isolation cost use of molecular cloning techniques can increase the concentration of protein in the cell
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how do you know if your purification is working? enzyme assay: necessary to determine the activity of the enzyme protein detection: determination of absolute amount of protein in the sample
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Specific Activity = activity of the enzyme in μmoles of product of the enzyme reaction per mg of protein in the solution. As purity goes up, the specific activity goes up. Everything else goes down. The protein becomes more pure as the purification proceeds Percent Recovery = amount of activity recovered as compared to the total amount of activity that was there to begin with. Can also report “Fold Purification” specific activity for that step compared to the original specific activity How do you know when the protein is pure? “…the demonstration, by all available methods, that the sample of interest consists of only one component.”
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Making a lysate—breaking open the cells Methods for breaking open the cells: osmotic lysis grinding using a blender homogenizer sonication—high frequency sound waves French Press—high pressure cell with a very small opening freeze-thaw cycles differential centrifugation
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In general: Bacteria, yeast, fungal and plant cells require more drastic methods than animal cells Methods for growing cells: fermentation techniques cell culture techniques animals, plants
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Stabilization of Proteins proteins have a characteristic 3D structure that needs to be maintained for activity low temperatures ~4 o C proteases and contaminating microorganisms will degrade the proteins buffers often include protease inhibitors, antimicrobials minimize handling, agitation prevent oxidation include antioxidants in buffers exclude metal ions work quickly
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Centrifugation techniques The faster you spin it, the more stuff you get rid of:
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Techniques for large volumes: Salt effects ionic strength: I = (0.5) Σ (c i Z i 2 ) where c i is the concentration of the i th ionic species in the solution, Z i is the charge on that species multiply charged species greatly increase the ionic strength of a solution protein solubility is a function of the protein concentration, the salt and the salt concentration
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solubility of carboxy-Hb in different salt solutions S = solubility in salt solution S’ = solubility in pure H 2 O consider what happens at low and at high salt concentrations increasing solubility at low salt concentrations decreasing solubility at high salt concentration, especially with divalent anions
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