Purification techniques


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Purification techniques

  1. 1. Protein Purification techniques
  2. 2. based on molecular size  - dialysis and ultrafiltration  - density gradient centrifugation  - size-exclusion chromatography based on solubility of proteins  - isoelectric precipitation  - salting out based on electric charge  - ion-exchange chromatography  - electrophoresis
  3. 3. Separation procedures based on molecular size  Procedures that separate proteins from small solutes.  Mermbane enclosing the protein solution is semipermeable.  Allows the exchange of water and small solutes (glucose, salts) that pass through the membrane freely but protein do not.  Molecules migrate through a semi permeable membrane under pressure or centrifugal force.
  4. 4. Density gradient (zonal) centrifugation  the rate of sedimentation is determined by weight, density and shape of macromolecule.. proteins in solution tend to sediment at high centrifugal fields.  in continuous density gradient of sucrose macromolecule sediment down at its own rate.
  5. 5. Chromatography Chromatography is a technique for separating mixtures into their components in order to analyze, identify, purify, and/or quantify the mixture or components. Separat e • Analyze • Identify • Purify • QuantifyComponent s Mixture
  6. 6. • Liquid Chromatography – separates liquid samples with a liquid solvent (mobile phase) and a column composed of solid beads (stationary phase) • Gas Chromatography – separates vaporized samples with a carrier gas (mobile phase) and a column composed of a liquid or of solid beads (stationary phase) Types of Chromatography
  7. 7. •Paper Chromatography – separates dried liquid samples with a liquid solvent (mobile phase) and a paper strip (stationary phase) • Thin-Layer Chromatography – separates dried liquid samples with a liquid solvent (mobile phase) and a glass plate covered with a thin layer of alumina or silica gel (stationary phase) Types of Chromatography
  8. 8. Principles of Paper Chromatography  Capillary Action – the movement of liquid within the spaces of a porous material is due to the forces of adhesion, cohesion, and surface tension. The liquid is able to move up the filter paper because its attraction to itself is stronger than the force of gravity.  Solubility – the degree to which a material (solute) dissolves into a solvent. Solutes dissolve into solvents that have similar properties. (Like dissolves like) This allows different solutes to be separated by different combinations of solvents.
  9. 9. Principles of Paper Chromatography Separation of components depends on both their solubility in the mobile phase and their differential affinity to the mobile phase and the stationary phase.
  10. 10. Column chromatography Chromatographic column (plastic or glass) include a solid, porous material (matrix) supported inside – stationary phase. A solution – the mobile phase - flows through the matrix (stationary phase). The solution that pass out of the bottom is constantly replaced from a reservoir. The protein solution migrates through column. They are retarded to different degrees by their interactions with the matrix material.
  11. 11. Gel-filtration chromatography Method uses porous particles to separate molecules of different size.  proteins passed over a column filled with a hydrated porous beads made of a carbohydrate or polyacrylamide polymer. mixture of proteins dissolved in suitable buffer, is allowed to flow by gravity down a column.  column is packed with beads of inert polymeric material.  very large molecules cannot penetrate into the pores of the beads, the small molecules enter the pores. large molecules are excluded and small proteins are retarded [large molecules exit (elute) first] .
  12. 12. Ion-exchange chromatography:  separation of proteins over a column filled with charged polymer beads (bead +charge =anion- exchange; bead -charge = cation exchange).  Positively charged proteins bind to beads of negative charge & vice versa. Bound proteins are eluted with salt. Least charged proteins will elute first.
  13. 13. Affinity chromatography:  proteins are passed through a column of beads containing a covalently bound high affinity group for the protein of interest. Bound protein is eluted by free high affinity group.  When a solution is passed the protein to be separated gets attached to substrate or enzyme.  It is then separated and obtained in pure form.
  14. 14. Affinity chromatography Example:  immunoaffinity chromatography: an antibody specific for a protein is immobilized on the column and used to affinity purify the specific protein.  Buffers containing a high concentration of salts and/or low pH are often used to disrupt the non covalent interactions between antibodies and antigen. A denaturing agent, such as 8 M urea, will also break the interaction by altering the configuration of the antigen-binding site of the antibody molecule.
  15. 15. High Pressure Liquid Chromatography (HPLC)  sample is vaporized and injected;  moves through a column containing stationary phase under high pressure;  separates mixture into compounds according to their affinity for the stationary phase  Short time procedure
  16. 16. 2. Separation procedures based on solubility  Isoelectric precipitation  Protein itself can be either positively or negatively charged overall due to the terminal amine -NH2 and carboxyl (-COOH) groups and the groups on the side chain.  Protein is positively charged at low pH and negatively charged at high pH. The intermediate pH at which a protein molecule has a net charge of zero is called the isoelectric point of that protein - pI 
  17. 17.  Protein is the least soluble when the pH of the solution is at its isoelectric point.  Different proteins have different pI values and can be separated by isoelectric precipitation
  18. 18. Salting out  Neutral salts influence the solubility of globular proteins.  Hydrophilic amino acid interact with the molecules of H2O, allow proteins to form hydrogen bonds with the surrounding water molecules.  Increasing salt concentration which decreases the number of water molecules available to interact with protein.  Increasing ionic strength decrease solubility of a protein.
  19. 19.  In general: a) small proteins more soluble than large proteins b) the larger the number of charged side chains, the more soluble the protein c) Proteins are usually least soluble at their isoelectric points.  Sufficiently high ionic strength completely precipitate a protein from solution.  Divalent salts [MgCl2, (NH4)SO4] are far more effective than monovalent (NaCl).
  20. 20. Separation procedures based on electric charge  Methods depend on acid-base properties, determined by number and types of ionizable groups of amino acids.  Each protein has distinctive acid-base properties related to amino acid composition.  Ionizing side chain groups:  R-COOH (Glu, Asp)  imidazole (His)  phenolic OH (Tyr)  e-amino (Lys)  guanidinyl (Arg)
  21. 21. Process of electrophoresis  1. sample application  2. adjustment of voltage or current - DIRECT CURRENT (gel-electrophoresis about 70 - 100 volts)  3. separation time: minutes (e.g. gel-electrophoresis of serum proteins 30 min.)  4. electrophoresis in supporting medium: fixation, staining and destaining  5. evaluation:  qualitative (standards)  quantitative (densitometry)
  22. 22. Electrophoretic method  negatively charged proteins move towards the anode  positively charged proteins move towards the cathode  much simple  much greater resolution  require small sample  Protein solution on the buffer (pH 8.6) is immobilized in a solid support (inert material like cellulose acetate)  Major protein components separate into discrete zones
  23. 23. Principle: Some substances have different net charges and can be separated into several fractions in external electric field. But velocity of a particle also depends on the: size, shape of the particle and given applied voltage
  24. 24. Serum proteins are separated into 6 groups: Albumin α1 - globulins α2 - globulins β1 - globulins β2 - globulins γ - globulins
  25. 25. Gel electrophoresis  Gel electrophoresis is a method that separates macromolecules (proteins, nucleic acids) on the basis of size, and electric charge.  Polyacryl amide or agarose gels are stabilizing media.  SDS (sodium dodecyl sulfate) – ionic surfactant, anionic substance.  Anions of SDS bind to peptide chain and protein is negatively charged, moves to cathode.
  26. 26. Principle: Serum proteins are negative charged at pH 8.6 (a buffer helps to maintain a constant pH) and they move toward the cathode at the rate dependent on their net charge. The separated proteins are fixed and stained by amidoblack solution.
  27. 27. Serum protein electrophoresis on agarose gel is a type of horizontal gel electrophoresis
  28. 28. The use of protein electrophoresis in diagnosis of diseases Acute inflammatory response • Immediate response occurs with stress or inflammation caused by infection, injury or surgical trauma • normal or albumin↓ • ↑ α1 and α2 globulins
  29. 29. Chronic inflammatory response • Late response is correlated with chronic infection (autoimmune diseases, chronic liver disease, chronic infection, cancer) • normal or albumin↓ •↑α1 or α2 globulins •↑↑ γ globulins
  30. 30. Liver damage - Cirrhosis • Cirrhosis can be caused by chronic alcohol abuse or viral hepatitis • ↓ albumin • ↓ α1, α2 and β globulins • ↑ Ig A in γ-fraction
  31. 31. Nephrotic syndrome • the kidney damage illustrates the long term loss of lower molecular weight proteins (↓ albumin and IgG – they are filtered in kidney) • retention of higher molecular weight proteins (↑↑ α2- macroglobulin and ↑β- globulin)
  32. 32. Monoclonal gammopathy  Monoclonal gammapathy is caused by monoclonal proliferation of β-lymphocytal clones. These „altered“ β-cells produce an abnormal immunoglobulin paraprotein.  Production of paraprotein is associated with benign monoclonal gammopathy (leukemia) and multiple myeloma.  Paraproteins can be found in a different position: between α-2 and γ-fraction.