Techniques%20in%20Protein%20Biochemistry

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Techniques in Protein Biochemistry

• Chapter 3

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• Problem isolation analysis of protein or
  amino acid found in cell
• Assumption can somehow analyze for wanted
  protein
• Common Colorimetric indicator (chemical rxn ?
  color formn can be monitored spectrophotometrica
  lly)
• Functional indicator (biological endpoint)
• This example colorimetric (breakdown of fats ?
  purple color)
• Activity assay
• Use at each step of separation

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Isolation of Wanted Protein from Brain Cells

• Brain cells contain wanted protein
• Open cells
• Homogenization, sonication, grinding
• Maintain cold, pH, osmolality
• Centrifugation often used Known speeds/
• conditions for
• diff organelles

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• Test each fraction for activity
• Save most active fractions
• Separation of wanted protein from other types of
  molecules
• Dialysis against physiological buffer

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Separation from other proteins

1. ChromatographyAll use solid or aqueous support
   to which wanted protein has some affinityAll
   use aqueous or gaseous mobile phase wanted
   protein has different affinityThis also moves
   molecules through/ past support

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• If wanted protein has greater affinity for
  support than for mobile phase, protein adheres
  to support phase
• If wanted protein has greater affinity for mobile
  phase than for support, protein will move with
  mobile phase through/away from support

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Gel Filtration Chromatography ( Size Exclusion)

• Separation by MW
• Solid support porous beads (ex sephadex,
  sepharose)
• Held in column
• Beads have microscopic pores/pits/spaces
• Mobile phase buffer of physio pH, ionic strength

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• Sample soln of wanted protein other
  (unwanted) proteins most have different MWs
• Apply sample to column
• Begin slow mobile phase flow
• Smaller proteins enter spaces in beads
• Larger proteins flow w/ buffer around beads (so
  emerge 1st from column)
• Collect fractions test each fraction by activity
  assay

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Ion Exchange Chromatography

• Separation by overall charge of proteins
• Solid support resin (charged microscopic beads)
  suspended in buffer
• Mobile phase buffer of particular pH, ionic
  strength
• Sample soln of wanted protein other
  (unwanted) proteins Most have different overall
  or charges of various strengths

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• Apply sample to column, begin slow mobile phase
  flow
• Proteins of charge opposite that of resin of
  similar strength of charge of resin Good
  affinity for resin bind electrostatically
• Proteins of the same charge or different strength
  of charge of resin No good affinity for resin
  flow through column quickly, so eluted first
• Result protein similar to resin is held in
  column

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• To elute protein held to resin in column use
  buffer of higher ionic strength or stronger pH
• Changes ionic environment
• Ions in new (elution) buffer exchange for
  protein (are more attractive to resin, so take
  the place of protein on resin)
• Collect fractions from mobile phase elution
  buffer
• Test all fractions by activity assay

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Affinity Chromatography

• Based on specificity of prot of interest for some
  molecules to which it alone will bind
• Ex Ab binds only specific Ag
• BUT binding must be reversible
• Solid support specific binding molecule
  (ligand) covalently bound to beads, etc.
• Mobile phase buffer of proper pH, ionic
  strength to maintain activity of prot of interest

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• Pack column apply sample
• Begin slow mobile phase flow
• Prot of interest ONLY will bind to ligand
• Types of binding (must be reversible) ionic,
  H-bonds, hydrophobic interactions
• To elute, may use solution of ligand (competes w/
  solid phase ligand) OR buffers of diff strength,
  pH that disrupt protein/ligand interactions

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Electrophoresis

• Separation AND identification
• Based on overall charge of protein ? movement
  under influence of electric field
• Zone
• Semisolid or gelatinous medium (plate or slab)
• Spot protein mixture (w/ wanted unwanted
  proteins in solution) onto gel
• Apply electric field

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• Molecules migrate toward anode ( charged)
• Distance traveled dependent on charge, size of
  protein Most impt size of protein
• Gel support acts as molecular sieve smaller
  molecules go faster toward anode, so migrate
  further
• Also, those more strongly charged move closer to
  anode
• Use chemical to stain aas ? bands representing
  proteins of decreasing MW

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• Run stds simultaneously
•  Mixture of proteins of known MW spot on one or
  several lanes
• Electrophorese under same conditions as unknown
  protein mixture
• Stain ? ladder of bands (lowest to highest MW
  proteins traveling some distance under these
  conditions)

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• Determine distance traveled for each band from
  origin
• Plot x distance migrated for each std of
  known MW y log MW of stds
• Yields std curve find distance traveled by
  unknown prot(s) on curve deter MW
• Can also cut gel, dissolve to free proteins

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Moving Boundary IsoElectric Focusing (IEF)

• Separation due to charge based on isoelectric pt
  of each prot
• Use gel of ampholytes (gel has regions of
  different pHs)
• Spot sample in middle of gel
• Apply electric field

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• Each prot in mixture will migrate toward or
  electrode, according to charge
• Each prot will stop moving when it reaches pH
  region of gel its isoelectric pH
• Stain aas of prots w/ chemical

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• Run stds simultaneously
• Mixture of proteins of known pIs spot in one or
  several lanes
• Electrophorese under same conditions as unknown
  protein mixture
• Stain ? ladder of bands (lowest to highest MW
  proteins traveling some distance under these
  conditions)
• Determine distance traveled for each band from
  origin
• Plot x distance migrated for each std of
  known pI
• y pH
• Yields std curve can find distance traveled by
  unknown prot(s) on curve ? pI                   
                                                   

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Characterize wanted protein by aa sequence

• Once wanted protein has been isolated from all
  other cell molecules
• Old method Break all peptide bonds ? solution
  of aas
• Analyze aas by chromatography
• Thin Layer Chromatography (TLC) on coated plate
  or paper support various mobile phases separate
  aas from each other
• High Pressure Liquid Chromatography (HPLC)
  force sample through small column packed with
  various types of support various mobile phases
  are forced through column by high pressure pumps
  to separate aas from each other

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• Now have identified all aas in protein
• With original protein, use chem. rxn to label
  amino terminal aa
• Use various enzs to cleave prot at partic aas
  along peptide chain ? peptide fragments
• Analyze fragments for overlap use knowledge of
  all aas in protein ? sequence

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• New method Automated
•   Chem rxn to label amino terminal aa
•   Cleave amino terminal aa
• Analyze for identity of last aa
• Rest of prot now has diff amino terminal aa
  (second to last in original prot)
• Chem rxn to label second to last aa of amino
  terminal
• Cleave this terminal aa
• Analyze for identity of second-to-last aa
• Etc. etc. etc.

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• Common now identify gene for protein of
  interest
• Isolate mRNAs (found w/in cell rich in wanted
  protein) w/ gene nucleotide sequence
• Use mRNAs to identify gene
• mRNA will have complimentary sequence to gene in
  DNA, so will pair in that region of DNA only
• Analyze gene for nucleotide sequence
• Use genetic code to determine aa sequence of
  wanted protein from gene which codes for it

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