Ch. 5. Protein Purification and Characterization Techniques

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Ch. 5. Protein Purification and Characterization Techniques Powered By Docstoc
					    Mary K. Campbell
    Shawn O. Farrell

       Chapter Five
  Protein Purification and
Characterization Techniques

 Paul D. Adams • University of Arkansas
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 (NH4SO4) commonly used to “salt
• 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
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
• Separates molecules based on size.
• Stationary phase composed of cross-linked gel
• 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- 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?
1) Determine which amino acids are present (amino
   acid analysis)
2) Determine the N- and C- termini of the sequence
   (a.a sequencing)
3) 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:
1) Enzymes- Trypsin, Chymotrypsin
2) Chemical reagents- Cyanogen bromide

Trypsin- Cleaves @ C-terminal of (+) charged side
Chymotrypsin- Cleaves @ C-terminal of aromatics
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
• 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

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