TAILIEUCHUNG - Lecture Principles of biochemistry - Chapter 3 (part 2): Protein purification and analysis

In this chapter 3 (part 2), you will: Know why it is important to purify a protein, know what organelles are in the pellet/supernatant during the differential centrifugation of a cell extract, understand how gel permeation chromatography works and what you can learn from this analysis, understand how ion exchange chromatography works,. | Chapter 3 (part 2) Protein purification and Analysis Why purify proteins? Pure proteins are required to study enzyme function Pure proteins are required for structural analysis (x-ray crystallography, NMR spectroscopy) Pure proteins are required to obtain amino acid sequence Steps in protein purification Develop assay Choose source of protein Prepare tissue extract cell disruption subcellular fractionation Protein fractionation (several steps) Determination of purity Differential Centrifugation tissue homogenate 1000 g Pellet unbroken cells nuclei chloroplast transfer supernatant transfer supernatant transfer supernatant 10,000 g 100,000 g Pellet mitochondria Pellet microsomal Fraction (ER, golgi, lysosomes, peroxisomes) Super. Cytosol, Soluble enzymes Chromatography Gel Permeation Chromatography + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + - - - - - - - - - +++ +++ +++ +++ +++ +++ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + - - - - - - - - - Cl- Cl- Cl- Cl- Cl- Cl- + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + - - - - - - - - - +++ +++ +++ +++ +++ +++ low salt buffer high salt buffer Ion-exchange Chromatography Affinity Chromatography Add excess ligand SDS poly acrylamide electrophoresis (PAGE) SDS = H3C-(CH2)10-CH2-OSO3- - - - - - - - - - - - - - - - - SDS – denatures protein coats w/ negative charge Used to determine protein MW And purity of protein prep Isoelectric Focusing Decreasing pH + - Decreasing pH + - pH 9 pH 3 2-D Electrophoresis Decreasing MW small large Decreasing pH + - SDS-PAGE Decreasing pH Decreasing MW Amino Acid Analysis Acid hydrolyze protein Treat with phenylisothiocyanate (PICT) Separate derivatized AA’s by HPLC + Protein Sequencing (Edman Degradation) + Trifluoroacetic acid 1) 2) 3) Repeat Can sequence in 30 to 60 AA’s from N-terminus Generate Proteolytic Fragments Endopeptidases Typsin cleaves at COOH end of Lys and Arg Chymotrypsin cleaves at COOH end of Phe, Tyr, Trp Chemical Cleavages Cyanogen Bromide cleaves at COOH end of Met Generate overlapping fragments Sequence individual fragments and piece together sequence Peptide mapping exercise Met-Ala-Arg- Gly-Glu-Tyr-Met-Cys-Lys-Phe-Ala-Glu-Gln-Asp Trypsin Met-Ala-Arg Phe-Ala-Glu-Gln-Asp Gly-Glu-Tyr-Met-Cys-Lys Chymotrysin Met-Ala-Arg- Gly-Glu-Tyr Met-Cys-Lys –Phe Ala-Glu-Gln-Asp CNBr Met Ala-Arg-Gly-Glu-Tyr-Met Cys-Lys-Phe-Ala-Glu-Gln-Asp Proteomic Analysis Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF)

• Sinh học
• Principles of biochemistry
• Lecture Principles of biochemistry
• Biological systems
• Biochemical compounds
• Protein purification
• Cell extract
• Ebook Principles of biochemistry
• Principles of biochemistry (4th edition)
• The glyoxylate cycle
• Fatty acid catabolism
• Electron Transfer reactions
• Evolutionary foundations
• Protein interactions modulated
• Nucleic acid structure
• Biological macromolecules
• Chemical principles
• Properties of water
• Hydrogen bonding
• Amino acids
• Amino acid structure
• Protein classification
• Protein 3 Dimensional structure
• Protein folding
• Protein 3 D structure
• 3 D structure
• Oxygen binding curve
• Enzyme kinetics
• Rate constants
• Catalytic Power
• Classes of enzymes
• Inhibitor kinetics
• Enzyme regulation
• Allosteric regulation
• Enzyme action
• Mechanisms of enzyme action
• Ions Cofactors
• Metal ion
• Nguyên lý hóa sinh
• Biotin cofactor
• Conformation of monosaccharides
• Carbohydrate classifications
• Oligo carbohydrate
• Polysaccharides carbohydrate
• Lipid subclasses
• Acyl lipids
• Fluid mosaic model
• membrane transporters
• Metabolism proceed
• Anaeorbic process
• Triose Phosphate Isomerase
• Regulation of glycolysis
• Control points in glycolysis
• Citric acid cycle
• TCA cycle