Chromatography,

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Chromatography,

  1. 1. Chromatography M.Prasad Naidu MSc Medical Biochemistry, Ph.D,.
  2. 2. Chromatography Components stationary phase (eg., solid matrix) mobile phase (eg., solvent) solute Solutes which interact differently with the stationary phase can be separated.
  3. 3. Apply Sample Continue Developing with Solvent
  4. 4. Common Media Used in Liquid Protein Chromatography Media Type Discrimination Ion Exchange Charge Gel Filtration Size and Shape Hydrophobic Surface Hydrophobicity Reverse Phase Total Hydrophobicity Affinity Specific Amino Acids Adsorption Amino Groups?
  5. 5. Chromatography Generic Protocol 1. Prepare Column () 2. Apply Sample 3. Wash 4. Elute 5. Analyze Fractions Equipment • batch-wise • home made • work stations • FPLC/HPLC HPLC = High Performance (Pressure) Liquid Chromatography FPLC = Fast Protein Liquid Chromatography
  6. 6. Ion Exchange Chromatography (IEC) • based on charge-charge interactions between solid matrix and solute
  7. 7. Basic Principal of IEC
  8. 8. increasing formate ion concentration 
  9. 9. Amino a. pKa Asp, Glu 4.3-4.7 His  7 Lys, Arg > 10 • Prepare or purchase column • Adjust pH and initial counter ion • Apply sample
  10. 10. Elution from IEC Column • change pH • increase counter-ion (ie, salt) concentration • in steps (eg, 0.1, 0.2, 0.3, 0.4 M NaCl) • gradually (eg, 00.4 M NaCl) with gradient maker
  11. 11. • collect fractions as column elutes • analyze fractions for components of interest
  12. 12.  increasing salting out effect anions: PO4, SO4, Cl, Br, NO3, Cl04, I, SCN cations: NH4, Rb, K, Na, Li, Mg, Ca, Ba increasingchaotropic effect Hydrophobic Interaction Chromatography (HIC) • separates proteins based on differences in hydrophobicity • absorb proteins to hydrophobic matrix • high salt promotes hydrophobic interactions • eg, 1 M (NH4)2SO4
  13. 13. HIC vs RPC Mobile Phase Polar Solvent Nonpolar Solvent Conditions Native Denatured Solute Discrimination Surface Residues Total Residues Reverse Phase Chromatography • separation based on total hydrophobicity • generally used to separate small peptides
  14. 14. Gel Filtration • separation based on size, aka • molecular sieve chromatography • size exclusion chromatography • media composed of cross- linked polymers • ‘pore’ size of matrix determines degree of interaction • larger molecules are excluded and migrate faster • smaller molecules are included and are retained longer  Dextran (=Sephadex®)  Agarose (=Sepharose®)  Polyacrylamide
  15. 15. Sephadex Code Range (kDa) G-25 1-5 G-50 2-30 G-100 4-150 G-150 5-300 G-200 5-600 • choose matrix with desired characteristics • size range • does not interact with solute • include 0.15-1 M NaCl in buffer • load sample in smallest possible volume • elute in one column volume Practical Considerations Applications: • purification • desalting • size determination
  16. 16. Calculating Size Vo = void volume Vt = total volume Ve = elution volume Ve - Vo Vt - Vo Kav = • use size standards • (relative MW) • migration also affected by shape
  17. 17. Affinity Chromatography • based on specific binding of protein to “ligand” • ligands can include: • substrate analogs, inhibitors • natural ligands • co-factors, metals • binding proteins • antibodies • Elution: destabilize binding • compete with free ligand • change pH, ionic strength • chaotropic or denaturing agents

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