Chapter 6. Techniques of Protein and Nucleic Acid Purification

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1 Chapter 6 Techniques of Protein and Nucleic Acid Purification

2 Considerations in protein expression and purification Protein source Natural sources Recombinant sources Methods of lysis and solubilization Osmotic, enzymatic or mechanical lysis Native or denaturing condition Stablilization of protein ph, temperature, protease, concentration, microorganism Assay of protein Enzymatic assay, binding assay, immunochemical assay

3 Enzyme-linked immunosorbent assay (ELISA)

4 General strategies of protein purification Solubility Salting in Salting out Ionic charge Ion exchange chromatography Electrophoresis Isoelectric focusing Polarity Adsorption chromatography Paper chromatography Reverse-phase chromatography Hydrophobic interaction chromatograph Molecular size Dialysis and ultrafiltration Gel electrophoresis Gel filtration chromatography Ultracentrifugation Binding specificity Affinity chromatography

5 Salting in: At low ionic strength, the protein solubility generally increases with the salt concentration (charge shielding) Salting out: At high ionic strength, the protein solubility generally decreases with the salt concentration (competition) Salting in and out Solubility of carboxy-hemoglobin at its isoelectric point

6 Fractionation by salting out

7 Isoelectric precipitation Solubility of a protein is lowest at its pi

8 Crystallization

9 Ion Exchange Chromatography Charged molecules bind to opposite charged groups on the column Anion exchange (DEAE) Cation exchange (CM) Affinity depends on the salt concentration and the ph

10 Ion Exchange Chromatography

11 Gel Filtration Chromatography (Size Exclusion Chromatography) The beads contain pores The buffer runs along the beads Small molecules can enter pores Large molecules cannot enter pores Larger molecules move faster

12 Gel Filtration Chromatography (Size Exclusion Chromatography)

13 Molecular mass determination by gel filtration chromatography

14 Molecular filtration: Dialysis and Ultrafiltration

15 Affinity chromatography Column contains a specific ligand Mixture of proteins runs through the column Proteins with affinity for the ligand stay behind Elution: free ligand, change of ph, salt concentration Good purity is generally obtained in a single step

16 Hydrophobic Interaction Chromatography Column contains hydrophobic groups (phenyl or octyl) Non-polar patches on proteins are excluded from the solvent Interactions are increased in high salt Elution with low salt buffer, detergents or changes in ph

17 High Performance Liquid Chromatography (HPLC) High resolution Fast High sensitivity Automation

18 Electrophoresis Separation by an electric field Polyacrylamide Gel Electrophoresis (PAGE) Agarose Gel Electrophoresis Capillary Electrophoresis Detection methods Staining Autoradiography Western, Southern or Northern blot

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20 SDS-PAGE Protein SDS SDS denatures proteins and effectively masks intrinsic charges of proteins, leading to identical charge-to-mass ratios and similar shapes

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22 Staining of protein gels Coomassie blue Silver staining

23 Staining of nucleic acid gel Ethidium bromide SYBR

24 Immunoblot (Western blot)

25 Two-dimensional (2D) gel electrophoresis

26 Capillary electrophoresis (CE) Electrophoresis in thin capillary tubes High voltage Rapid and sharp separation High resolution and automation Small amount of sample DNA sequencer

27 Ultracentrifugation Macromolecules sediment under enormous accelerations Sedimentation rate varies with mass and shape of a protein, density of the medium Sedimentation coefficient (S) Analytical or preparative

28 Sedimentation coefficient

29 Zonal ultracentrifugation

30 Isopycnic ultracentrifugation