Chromatography

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Chromatography

• Desalting and Affinity

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Chromatography

• Technique to separate components of a mixture by
  passing them through a matrix.
• A solvent is used for carrying sample mixture
  through the matrix
• Separation occurs because each compound in a
  mixture interacts differently with the matrix.

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Gel-Filtration

• Ammonium sulfate interferes with affinity
  chromatographic step.
• We have to desalt our extract by using either
  dialysis or gel filtration.
• Gel filtration chromatography is a separation
  based on size. It is also called molecular
  exclusion or gel permeation chromatography.

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Gel-Filtration

• Separation mechanism is not based on adsorption.
• It is independent of the eluent system
• Has high recoveries both on basis of mass and
  biological activity.
• Since it is iso-cratic, samples are diluted.

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Gel- Filtration

• The volume between the beads is called as void
  volume accessible to all molecules.
• The volume in the pore is the pore volume
• The non-porous part of the beads is the backbone
  volume not accessible to samples.
• Partition occurs between the molecules that have
  access to the void volume and molecules that have
  access to void and pore volume.

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Gel-Filtration

• Three steps
• Equilibration
• Sample loading
• Elution

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Picture from Amersham website

• Pores in the gel

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Desalting

• Group Separation Mode Used in separating small
  molecules from large molecules in a mixture
• Macromolecules have access only to the void
  volume and therefore grouped and eluted together.
• Small molecules (neutral salts, buffer salts, low
  mw additives) are separated as a single unit as
  late as possible. They have access to pore
  volume.
• The large difference in the elution volumes allow
  sample volumes up to 30 of the column volume.
• High flow rates can be applied and broad or
  narrow columns can be used.

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Sephadex

• Sephadex -50 is the matrix which exploits both
  physical and chemical properties of the mixture.
• These highly specialized gel filtration and
  chromatographic media are composed of macroscopic
  beads synthetically derived from the
  polysaccharide, dextran(polymer of glucose).
• The organic chains are cross-linked to give a
  three dimensional network having functional ionic
  groups attached by ether linkages to glucose
  units of the polysaccharide chains.
• Available forms include anion and cation
  exchangers, as well as gel-filtration resins,
  with varying degrees of porosity bead sizes fall
  in discrete ranges between 20 and 300µ.

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http//web.siumed.edu/bbartholomew/images/protein
_methods/sephadex.gif
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Affintiy Chromatography

• Affinity chromatography (AC) is a technique
  enabling purification of a biomolecule with
  respect to biological function or individual
  chemical structure.
• The substance to be purified is specifically and
  reversibly adsorbed to a ligand (binding
  substance), immobilized by a covalent bond to a
  chromatographic bed material (matrix).
• Samples are applied under favourable conditions
  for their specific binding to the ligand.
• Substances of interest are consequently bound to
  the ligand while unbound substances are washed
  away.
• Recovery of molecules of interest can be achieved
  by changing experimental conditions to favour
  desorption. AC media are commonly used for
  applications such as purification of fusion
  proteins, mono- and polyclonal antibodies, and
  glycoproteins.

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Affinity Chromatography (AC)

• AC involves an inert matrix coupled with a
  affinity ligand specific for a binding site on
  the target molecule.
• Under suitable binding conditions this affinity
  matrix will bind molecules according to its
  specificity only.
• All other sample components will pass through the
  medium unadsorbed.

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Affinity Chromatography

• After washing, condition are changed to
  disassociate or displace the molecules from the
  ligand.
• simple "on-off" mode of chromatography is applied
  by switching abruptly from full binding to
  complete release conditions.
• Thus AC fishes out the target molecule by way of
  highly specific binding and release, rather than
  removing contaminants by fine-tuned isocratic or
  gradient elution techniques.

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Affinity Chromatography

• Two important things to consider
• Finding a ligand specific enough to allow step
  elution.
• Finding conditions for safe binding and release
  within the stability window of the target
  molecule and the ligand.

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Affi-Gel Blue

• Agrose gel covalently attached Cibacron Blue F3GA
  dye.
• 1.9mg dye per mL of gel
• We are using 50-100 mesh (150-300 µm)
• Separates nucleotide dependent enzymes
  (dinucleotide fold biospecifically bind to the
  dye)
• Enzyme can be eluted from the dye with a specific
  nucleotide co-factor

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Sigma-Aldrich site