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Chromatography

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					Chromatography

Desalting and Affinity
           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.
              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.
             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.
              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.
             Gel-Filtration
• Three steps
  – Equilibration
  – Sample loading
  – Elution
Picture from Amersham website
• Pores in the gel
                        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.
                   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.
http://web.siumed.edu/~bbartholomew/images/protein_methods/sephadex.gif
        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.
  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.
      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.
     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.
              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
Sigma-Aldrich site

				
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posted:7/23/2011
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