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Lecture 6 Working with proteins

Lecture 6 Working with proteins. Protein purification. Source of protein Overexpression of protein Fractionation procedures Solubility Salting out ((NH 4 ) 2 SO 4 ) pH changes Size Centrifugation Size exclusion (gel filtration) Charge/polarity Ion exchange chromatography

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Lecture 6 Working with proteins

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  1. Lecture 6Working with proteins

  2. Protein purification • Source of protein • Overexpression of protein • Fractionation procedures • Solubility • Salting out ((NH4)2SO4) • pH changes • Size • Centrifugation • Size exclusion (gel filtration) • Charge/polarity • Ion exchange chromatography • Hydrophobic exchange chromatography • Other • Affinity chromatography

  3. Size-exclusion Chromatography

  4. Separation base on size 1. Zonal centrifugation Based on sedimentation coefficient (s) m = mass of protein (1-v) =buoyancy factor m(1-v) s= f f = frictional coefficient Spin in centrifuge Separation according to sedimentation coefficient Layer sample on top Tube with density gradient Particles of higher M.W and density will be closer to bottom of the tube

  5. Ion Exchange Chromatography Example: DEAE - Cellulose (Diethylamino Ethyl) Anion exchange:(+) charged resin, binds (-) proteins Cation exchange:(-) charged resin, binds (+) proteins

  6. Ion Exchange Chromatography 1.Bind protein to column in low salt buffer 2.Wash column 3.Elute protein with high salt buffer. Fractions

  7. Ion Exchange Chromatography Salt Protein Activity 2 4 6 8 10 12 Fraction

  8. Affinity Chromatography See Fig 6-2 (b)

  9. Polyhistidine tag purification Genetically modified protein with 6 or more histidines at C or N-terminal end Nickel-containing column Elute with imidazole Pure protein with imidazole (Can be removed by dialysis)

  10. Gel electrophoresis m =electrophoretic mobility =velocity of particle z  E=electric field strength z=net charge on protein f=frictional coefficient = m= E f Net charge on protein comes from SDS (negatively charged detergent) Proteins are separated on the basis of mass (frictional coefficient) SDS disrupts almost all noncovalent interactions SDS binds to protein 1 SDS molecule per 2 amino acids

  11. O H NH2 C C H2C Gel electrophoresis Polyacrylamide gel formation O H ) ( CH2 NH C C H2C 2 + acrylamide methylenebisacrylamide S2O82- SO4-· sulfate free radical H H H C H2C C C H2C H2C Pore size can be controlled by adjusting the concentration of the monomer and crosslinker CO NH CO NH2 CO NH2 CH2 CO NH H H C H2C C C H2C H2C H CO NH2 CO NH2

  12. Electrophoresis Log(MW) Relative mobility

  13. 2kT (c2/(c1(r12-r22)) m = ln (1-v)2 Analytical ultracentrifugation Same principle as zonal centrifugation Spin at a low speed until sedimentation rate is counterbalanced by diffusion rate Smooth gradient of protein develops Measure conc.(c1,c2) at different points(r1,r2) from center of rotor Very precise way to determine MW

  14. Isoelectric focusing - Decreasing pH (stable pH gradient) + Apply protein to top Apply electric field protein migrates to pH where its net charge is zero (pI)

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