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Campbell6e lecture ch5

Campbell6e lecture ch5






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    Campbell6e lecture ch5 Campbell6e lecture ch5 Presentation Transcript

    • Chapter Five Protein Purification and Characterization Techniques
    • Isolation of Proteins from Cells
      • Many different proteins exists within one cell
        • • Many steps needed to extract protein of interest, and separate from many contaminants
        • • Before purification begins, protein must be released from cell by homogenization
    • How We Get Proteins Out of Cells
    • Salting Out
      • • After Proteins solubilized, they can be purified based on solubility (usually dependent on overall charge, ionic strength, polarity)
      • • Ammonium sulfate (NH 4 SO 4 ) commonly used to “salt out”
      • • Takes away water by interacting with it, makes protein less soluble because hydrophobic interactions among proteins increases
      • • Different aliquots taken as function of salt concentration to get closer to desired protein sample of interest ( 30, 40, 50, 75% increments )
      • • One fraction has protein of interest
    • Differential Centrifugation
      • • Sample is spun, after lysis, to separate unbroken cells, nuclei, other organelles and particles not soluble in buffer used
      • • Different speeds of spin allow for particle separation
    • Column Chromatography
      • • Basis of Chromatography
        • Different compounds distribute themselves to a varying extent between different phases
      • • Interact/distribute themselves
      • • In different phases
      • • 2 phases:
      • • Stationary : samples interacts with this phase
      • • Mobile : Flows over the stationary phase and carries along with it the sample to be separated
    • Column Chromatography
    • Size-Exclusion/Gel-Filtration
      • • Separates molecules based on size.
      • • Stationary phase composed of cross-linked gel particles.
      • • Extent of cross-linking can be controlled to determine pore size
      • • Smaller molecules enter the pores and are delayed in elution time. Larger molecules do not enter and elute from column before smaller ones.
    • Size Exclusion/Gel-filtration (Cont’d)
    • Affinity Chromatography
      • • Uses specific binding properties of molecules/proteins
      • • Stationary phase has a polymer that can be covalently linked to a compound called a ligand that specifically binds to protein
    • Ion Exchange
      • • Interaction based on overall charge (less specific than affinity)
      • • Cation exchange
      • • Anion exchange
    • Electrophoresis
      • • Electrophoresis - charged particles migrate in electric field toward opposite charge
      • • Proteins have different mobility:
        • Charge
        • Size
        • Shape
      • • Agarose used as matrix for nucleic acids
      • • Polyacrylamide used mostly for proteins
    • Electrophoresis (Cont’d)
      • • Polyacrylamide has more resistance towards larger molecules than smaller
      • • Protein is treated with detergent (SDS) sodium dodecyl sulfate
      • • Smaller proteins move through faster (charge and shape usually similar)
    • Isoelectric Focusing
      • • Isolectric focusing- based on differing isoelectric pts. (pI) of proteins
      • • Gel is prepared with pH gradient that parallels electric-field. What does this do?
      • • Charge on the protein changes as it migrates.
      • • When it gets to pI, has no charge and stops
    • Primary Structure Determination
      • How is 1˚ structure determined?
      • Determine which amino acids are present (amino acid analysis)
      • Determine the N- and C- termini of the sequence (a.a sequencing), and the Internal Residues
      • Determine the sequence of smaller peptide fragments (most proteins > 100 a.a)
      • 4) Some type of cleavage into smaller units necessary
    • Primary Structure Determination
    • Protein Cleavage
      • Protein cleaved at specific sites by:
      • Enzymes- Trypsin, Chymotrypsin, Carboxypeptidases (C-terminus)
      • Chemical reagents
      • Cyanogen bromide, cleaves at Methionine;
      • PITC, cleaves from N-terminus (Edman Degradation)
      • Hydrazine, cleaves from C-terminus
      • Enzymes which cleaves Internal Residues:
      • Trypsin- Cleaves @ C-terminal of (+) charged side chains (basic amino acid)
      • Chymotrypsin- Cleaves @ C-terminal of aromat ics
    • Peptide Digestion
    • Cleavage by CnBr
      • Cleaves @ C-terminal of INTERNAL methionines
    • Determining Protein Sequence
      • After cleavage, mixture of peptide fragments produced.
      • • Can be separated by HPLC or other chromatographic techniques
      • • Use different cleavage reagents to help in 1˚ determination
    • Peptide Sequencing
      • • Can be accomplished by Edman Degradation
      • • Relatively short sequences (30-40 amino acids) can be determined quickly
      • • So efficient, today N-/C-terminal residues usually not done by enzymatic/chemical cleavage
    • Peptide Sequencing