Techniques of Protein Purification - PowerPoint PPT Presentation

slide1 n.
Download
Skip this Video
Loading SlideShow in 5 Seconds..
Techniques of Protein Purification PowerPoint Presentation
Download Presentation
Techniques of Protein Purification

play fullscreen
1 / 31
Techniques of Protein Purification
1474 Views
Download Presentation
yates
Download Presentation

Techniques of Protein Purification

- - - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript

  1. Chapter 6 Techniques of Protein Purification You should become familiar with this entire chapter so that you can use it for reference. But you are only responsible for the topics in the chapter that were covered in lecture.

  2. Protein isolation Selection of protein source • Tissues from animals • Microorganisms (E. coli or yeast) • Molecular cloning techniques Methods of solubilization • Osmosis lysis (with hypotonic solution) • Use of lysozyme (enzyme that degrades cell wall) • French press or sonication

  3. Stabilization of proteins • 1. pH (think buffers!) • 2. Temperature (close to 0oC) • Thermal stability could be used for purification • 3. Addition of protease inhibitors • 4. Gentle handling (no frothing) • Assay of proteins • 1. If purifying an enzyme, use the • reaction it catalyzes as an assay • 2. If metalloprotein use the metals to follow the protein • 3. Immunochemical techniques (antibodies)

  4. General strategy of protein purification Proteins are purified by fractionation procedures, a series of independent steps in which the properties of protein of interest are utilized to separate it from other contaminating proteins. How do we know our sample of protein is pure? We don't! The best we can do is to demonstrate by all available methods that our sample consists of only one component.

  5. General strategy of protein purification CharacteristicProcedure Solubility 1. Salting in 2. Salting out Ionic charge:1. Ion exchange chromatography 2. Electrophoresis 3. Isoelectric focusing Polarity: 1. Adsorption chromatography 2. Paper chromatography 3. Hydrophobic interaction chromatography Molecular size: 1. Dialysis and ultrafiltration 2. Gel electrophoresis 3. Gel filtration chromatography 4. Ultracentrifugation Binding specificity: 1. Affinity chromatography

  6. Solubility of a protein in aqueous solution Depends strongly on: Concentrations of dissolved salts pH Temperature 4. Addition of water-miscible organic solvents, e.g., ethanol or acetone

  7. Solubility of carboxyhemoglobin at its isoelectric point as a function of ionic strength and ion type Page 131

  8. Solubilities of several proteins in ammonium sulfate solutions Page 131

  9. Solubility of b-lactoglobin as a function of pH at several NaCl concentrations Page 132

  10. Isoelectric Points of Several Common Proteins Protein pI Pepsin 1.0 Ovalbumin (hen) 4.6 Serum albumin (human) 4.9 Tropomyosin 5.1 Insulin (bovine) 5.4 Fibrinogen (human) 5.8 g-Globulin (human) 6.6 Collagen 6.6 Myoglobin (horse) 7.0 Hemoglobin (human) 7.1 Ribonuclease A (bovine) 9.4 Cytochrome c (horse) 10.6 Histone (bovine) 10.8 Lysozyme (hen) 11.0 Salmine (salmon) 12.1

  11. Protein crystals Page 133

  12. Chromatographic separations Protein separation and purification by column chromatography From Lehninger Principles of Biochemistry

  13. Column Chromatography: Ion Exchange From Lehninger Principles of Biochemistry

  14. A schematic diagram illustrating the separation of several proteins by ion exchange chromatography using stepwise elution

  15. Column Chromatograph: Size-exclusion Gel filtration chromatography can be used to estimate molecular masses From Lehninger Principles of Biochemistry

  16. A schematic illustration of gel filtration chromatography

  17. The separation of macromolecules by affinity chromatography

  18. Hydrophobic interaction chromatography: Proteins contain hydrophobic amino acid side-chains, some of which are exposed at the surface of the protein. Proteins will therefore often bind to other hydrophobic molecules. Hydrophobic interaction columns are produced by covalently attaching hydrophobic molecules such as acyl chains or phenyl groups to insoluble carbohydrate resins.

  19. Dialysis is a form of molecular filtration

  20. Protein separation and characterization by Electrophoresis Migration of ions in an electric field is widely used for the analytical separation of biomolecules.

  21. Polymerization of acrylamide and N,N¢-methylenebisacrylamide to form a cross-linked polyacrylamide gel Page146

  22. SDS binds to proteins in amounts roughly proportional to molecular weight. • The high negative charge of the SDS-protein complex is greatly in excess of the inherent charge of the protein. • Electrophoresis in the presence of SDS therefore separates almost exclusively by molecular weight.

  23. Polyacrylamide gel • Stain proteins to visualize, e.g., Coomassie blue (see page 93) • Rate of migration depends roughly on charge-to-mass ratio. • (Protein shape may also influence migration rate.) Larger proteins move more slowly Smaller proteins move faster From Lehninger Principles of Biochemistry

  24. From Lehninger Principles of Biochemistry

  25. A logarithmic plot of the molecular masses of 37 different polypeptide chains ranging from 11 to 77 kD versus their relative electrophoretic mobilities on an SDS-polyacrylamide gel

  26. Detection of proteins by immunoblotting (aka Western Blot) Page 148

  27. Setting up a purification Table Total Enzyme Activity: the activity per ml of enzyme solution multiplied by the total volume of that fraction. Total Protein: the protein concentration per ml of solution multiplied by the total volume of that fraction. Specific activity: Total Activity/Total Protein or Activity/Protein concentration Fold-purification: (Specific activity at a given step)/(Specific activity of starting sample) Yield = (Total activity at a given step) / (Total activity of starting sample)*100

  28. Setting Up a Purification Table: Total Enzyme Activity: the activity per ml of enzyme solution multiplied by the total volume of that fraction. (If you have 50 ml of the original homogenate, the volume is 50 ml) Total Protein: the protein concentration per ml of solution multiplied by the total volume of that fraction. Specific activity: Total Activity/Total Protein or Activity/Protein concentration Fold-purification: (Specific activity at a given step)/(Specific activity of starting sample) Yield: (Total activity at a given step)/(Total activity of starting sample)*100

  29. From Lehninger Principles of Biochemistry

  30. Purification of Rat Liver Glucokinase Page 142