Chapter Five Protein Purification and Characterization Techniques - PowerPoint PPT Presentation

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Chapter Five Protein Purification and Characterization Techniques

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Chapter Five Protein Purification and Characterization Techniques Isolation of Proteins from Cells Many different proteins exists within one cell Many steps ... – PowerPoint PPT presentation

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Title: Chapter Five Protein Purification and Characterization Techniques


1
Chapter FiveProtein Purification and
Characterization Techniques
2
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

3
How We Get Proteins Out of Cells
4
Salting Out
  • After Proteins solubilized, they can be
    purified based on solubility (usually dependent
    on overall charge, ionic strength, polarity
  • Ammonium sulfate (NH4SO4) 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

5
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

6
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

7
Column Chromatography
8
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.

9
Size Exclusion/Gel-filtration (Contd)
10
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

11
Ion Exchange
  • Interaction based on overall charge (less
    specific than affinity)
  • Cation exchange
  • Anion exchange

12
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

13
Electrophoresis (Contd)
  • 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)

14
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

15
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)
  • Determine the sequence of smaller peptide
    fragments (most proteins gt 100 a.a)
  • 4) Some type of cleavage into smaller units
    necessary

16
Primary Structure Determination
17
Protein Cleavage
  • Protein cleaved at specific sites by
  • Enzymes- Trypsin, Chymotrypsin
  • Chemical reagents- Cyanogen bromide
  • Enzymes
  • Trypsin- Cleaves _at_ C-terminal of () charged side
    chains
  • Chymotrypsin- Cleaves _at_ C-terminal of aromatics

18
Peptide Digestion
19
Cleavage by CnBr
  • Cleaves _at_ C-terminal of INTERNAL methionines

20
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

21
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

22
Peptide Sequencing
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