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Protein Purification and Characterization

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High concentration of desired protein. Separation Procedures: ... Based on attraction between charged groups in protein and column medium(insoluble beads or gel) ... – PowerPoint PPT presentation

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Title: Protein Purification and Characterization


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Protein Purification and Characterization
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  • Strategy of Purification
  • Source
  • Readily available biological source.
  • High concentration of desired protein.
  • Separation Procedures
  • Based on different physical properties of
    proteins.
  • Apply several procedures in succession.
  • Analysis of Purification
  • Assay for desired protein and total protein.
  • Continue purification with fractions enriched in
    desired protein.

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Salting Out
  • Add salt to aqueous solution of protein.
  • As salt concentration increases, salt and protein
    compete for water, protein molecules aggregate
    and precipate.
  • Protein dissolves if salt is removed.
  • Salt of choice Ammonium sulfate pure, highly
    soluble, gentle.

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Ion Exchange Chromatography Based on
attraction between charged groups in protein and
column medium(insoluble beads or gel). Selective
absorption and elution accomplished by change of
pH or by change in salt concentration.
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Affinity Chromatography Based on
selective binding of ligand by binding protein
(substrate binding by enzyme or binding of
regulatory molecule to regulated enzyme). Ligand
covalently bound to insoluble medium (bead or
gel). Pass protein over column. Only binding
protein is absorbed. Elute with high
concentration of unbound ligand.
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Polyacrylamide Gel Electrophoresis (PAGE) Form
gel in apparatus by polymerizing acrylamide
with Crosslinker (bis-acrylamide) all in buffer
solution(defines pH of gel). Load protein
sample. Apply electrical voltage to separate
protein based on combination of charge and
shape. Detect protein by staining with Coomassie
Blue or silver stain or detect radioactivity with
X-ray film. Use Western blot with radioactive
antibody.
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X-ray Crystallography Crystallize
protein sample. Pass beam of X-ray through
crystal. Measure angles and intensities of
diffraction of X-rays. Compute electron density
maps. Make a three dimentional model.
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