Affinity chromatography
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Affinity chromatography

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    Affinity chromatography Affinity chromatography Presentation Transcript

    • AFFINITY CHROMATOGRAPHY By: H. Nur Halipçi
    • CHROMATOGRAPHY
      • Chromatography is the collective term for a set of lab orator y techniques for the separation of mixtures.
      • The term comes from Gree k χρώμα: chroma means color and γραφειν: graphein means write
    • INVENTED BY
      • 1903 – Tswett, a Russian botanist coined the term chromatography.
    • CHROMATOGRAPHY
      • Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary ( immobilize phase) while the other (the mobile phase) moves in a definite direction
      • An animation
      • http://www.dnatube.com/video/2895/Column-Chromatography
    • TERMINOLOGY
      • An immobilized phase is a stationary
      • phase which is immobilized on the support
      • particles, or on the inner wall of the column
      • tubing
    • TERMINOLOGY
      • The mobile phase is the phase which moves in a definite direction. It may be a liquid (LC ) , a gas (GC), or a supercritical fluid (SFC). A better definition:The mobile phase consists of the sample being s eparated and the solvent that moves the sample through the column.
    • TERMINOLOGY
      • The analyte is the substance that is to
      • be separated during chromatography.
    • TERMINOLOGY
      • The sample is the matter analysed in chromatography. It may consist of a single component or it may be a mixture of components.
    • TERMINOLOGY
      • E lution
      • - washing of the mixture
      • Eluent
      • - additional solvents used for elution
      • Residency
      • - time spent on column
    • Types of Chromatography
    • AFFINITY CHROMATOGRAPHY
    • Affinity History
      • 1930s, first developed by A . Wilhelm Tiselius -a swedish biochemist , won the Nobel Prize in 1948
      • Used to study enzymes and other proteins
      • Relies on the affinity of various biochemical compounds with specific properties
    • Examples
        • Antigen Antibody
        • Antibody Antigen
        • Substrate Enzyme
        • DNA Histon
        • Hormone Binding Protein/Receptor
    • Specificity of Affinity Chromatography
      • Specificity is based on three aspect of affinity
      • Matrix : for ligand attachment.
              • Spacer arm : used to bind ligand to matrix
              • Ligand : molecule that binds reversibly to a specific target molecule(site of interaction)
    • An animation www6.gelifesciences.com/aptrix/...nsf/.../ Affinity /.../GE_ Affinity .swf -
    • So now what….
      • The Sample is injected into the equilibrated affinity chromatography column
      • Only the substance with affinity for the ligand are retained on the column
      • The substance with no affinity to the ligand will elute off
      • The substances retained in the column can be eluted off by changing the pH of salt or organic solvent concentration of the eluent
    • Matrix
      • The matrix simply provides a structure to increase the surface area to which the molecule can bind
      • The matrix must be activated for the ligand to bind to it but still able to retain it’s own activation towards the target molecule
    • Matrix
      • Amino, hydroxyl, carbonyl and thio groups located with the matrix serve as ligand binding sites
      • Matrix are made up of agarose and other polysaccharides
      • The matrix also must be able to withstand the decontamination process of rinsing with sodium hydroxide or urea
    • Ligand
      • The Ligand binds only to the desired molecule within the solution
      • The ligand attaches to the matrix which is made up of an inert substance
      • The ligand should only interact with the desired molecule and form a temporary bond
      • The ligand/molecule complex will remain in the column, eluting everything else off
      • The ligand/molecule complex dissociates by changing the pH
    • Antibody affinity (Immunoaffinity Chromatography)
      • Used to purify antibody against a specific antigen
      • Ex:Immunoglobulins
      • P urification of IgG , IgG fragments and subclasses have the high affinity of protein A and protein G for the Fc region of polyclonal and monoclonal IgG-type antibodies
    • Protein A and protein G
      • Protein A and protein G are bacterial cell surface proteins (from Staphylococcus aureus and Streptococcus respectively)
      • Recombinant protein A is available;
      • Engineered to include a C-terminal
      • Results in an enhanced binding capacity
    • AFFINITY CHROMATOGRAPHY
      • Can be used;
      • Purify and concentrate a substance from a mixture into a buffering solution
      • Reduce the amount of a substance in a mixture
      • Discern what biological compounds bind to a particular substance, such as drugs
      • Purify and concentrate an enzyme solution
    • Applications
      • Used in Genetic Engineering
      • - nucleic acid purification
      • Production of Vaccines
      • - antibody purification from blood serum
      • And Basic Metabolic Research
      • - protein or enzyme purification from cell free extracts
      • Avidin(or strepdavidin) -biotin interaction is used to purify proteins
      • Avidin :protein deposited in the whites of eggs(birds,reptiles…)
      • Streptavidin is a tetrameric protein purified from the bacterium Streptomyces avidinii
      • Biotin: ( vitamin H or B7) cofactor in the metabolism of fatty acids and leucine, and in gluconeogenesis
      PROTEIN PURIFICATION
      • The non-covalent bond formed between biotin and avidin or streptavidin
      • has a binding affinity >most antigen and antibody bonds
      • ~ strength of a covalent bond
    • biotinylation
      • means affinity chromatography using immobilized avidin or streptavidin
      • to separate the biotinylated protein from a mixture of other proteins and biochemicals
    • Nucleic acid separation using immobilized metal affinity chromatography (IMAC)
      • The method can be used to purify compounds
      • containing purine or pyrimidine moieties where
      • the purine and pyrimidine moieties are shielded
      • from interaction with the column matrix from
      • compounds containing a non-shielded purine or
      • pyrimidine moiety or group.
      • Thus, double-stranded plasmid and genomic DNA, which has no low binding affinity can be easily separated from RNA or oligonucleotides which bind strongly to metal-charged chelating matrices
      • IMAC columns clarify plasmid DNA from bacterial alkaline lysates, purify a ribozyme, and remove primers and other contaminants from PCR reactions
    • ADVANTAGES OF AFFINITY CHROMATOGRAPHY
      • Extremely high specificity
      • High degrees of purity can be obtained
      • The process is very reproducible
      • The binding sites of biological molecules can be simply investigated
    • DISADVANTAGES OF AFFINITY CHROMATOGRAPHY
      • 1) Expensive ligands
      • 2) Leakage of ligand
      • 3) Degradation of the solid support
      • 4) Limited lifetime
      • 5) Non-specific adsorption
      • 6) Relatively low productivity
    • REFERENCES
      • [1]http://en.wikipedia.org/wiki/Affinity_chromatography
      • [2] www.apsu.edu/reedr/.../ Affinity %20 Chromatography %201.ppt
      • [3] www.rpi.edu/dept/chem-eng/WWW/faculty/.../Lecture%2001.pdf -
      • [ 4] www.chemistryinnovation.co.uk/.../Technology%20Area%20 Affinity %20 Chromatography .pdf -
      • THANKS…