5-Notes-ProteinTechniques

5-Notes-ProteinTechniques - Chapter 3 Berg Exploring...

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Unformatted text preview: Chapter 3 -- Berg Exploring Proteins and Proteomes Techniques to study proteins • Protein purifcation techniques • How do you know the amino acid sequence oF a protein? • Immunological techniques to study proteins • In-vitro protein synthesis • Mass spectrometry • X-ray crystallography and NMR 1 Wednesday, October 19, 2011 Chromatography 2 Wednesday, October 19, 2011 Gel-fltration chromatography or size-exclusion chromatography 3 Wednesday, October 19, 2011 Gel-fltration chromatography 4 Wednesday, October 19, 2011 Ion-exchange chromatography 5 Wednesday, October 19, 2011 6 Wednesday, October 19, 2011 Ion-exchange chromatography 7 Wednesday, October 19, 2011 AfFnity chromatography 8 Wednesday, October 19, 2011 9 Wednesday, October 19, 2011 10 Wednesday, October 19, 2011 Gel electrophoresis 11 Wednesday, October 19, 2011 12 Wednesday, October 19, 2011 13 Wednesday, October 19, 2011 14 Wednesday, October 19, 2011 15 Wednesday, October 19, 2011 Isoelectric focusing depends on the pI of proteins 16 Wednesday, October 19, 2011 2D-electrophoresis 17 Wednesday, October 19, 2011 18 Wednesday, October 19, 2011 Protein purifcation • How do you know the protein is there? How do you recognize it? • How do you puriFy it From others? • How do you evaluate your purifcation scheme? • How do you know the size oF a protein? 19 Wednesday, October 19, 2011 How do you evaluate your purifcation scheme? • Total protein • Total activity • Specifc activity • Yield • Purifcation Level 20 Wednesday, October 19, 2011 21 Wednesday, October 19, 2011 Protein purifcation • How do you know the protein is there? How do you recognize it? • How do you puriFy it From others? • How do you evaluate your purifcation scheme? • How do you know the size oF a protein? 22 Wednesday, October 19, 2011 How do you know the size of a protein? • Zonal centrifugation (sedimentation coefFcient) • SDS-PAGE (electrophoresis) • Mass Spectroscopy 23 Wednesday, October 19, 2011 Wednesday, October 19, 2011 Wednesday, October 19, 2011 26 Wednesday, October 19, 2011 27 Wednesday, October 19, 2011 28 Wednesday, October 19, 2011 29 Wednesday, October 19, 2011 Sedimentation coefFcient • s = m (1 - ν ρ )/f • s = sedimentation coefFcient • m= mass • ν = partial speciFc volume • ρ = density of the medium • f = frictional ratio 30 Wednesday, October 19, 2011 Mass spectroscopy 31 Wednesday, October 19, 2011 32 Wednesday, October 19, 2011 33 Wednesday, October 19, 2011 To know the amino acid sequence of a protein is useful Similarity among proteins (classes) Evolution of proteins Existence of repeats or modules Identify localization signals Prepare antibodies against a protein Allow the design of DNA probes to locate a gene 34 Wednesday, October 19, 2011 Chapter 3 -- Berg Exploring Proteins and Proteomes How do you isolated a protein from others?...
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5-Notes-ProteinTechniques - Chapter 3 Berg Exploring...

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