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    Affinity Chromatography: Techniques in Biochemistry

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    Sadia Din
    Affinity chromatography is a separation and purification method that exploits the biological affinity between a protein and ligand coupled to a chromatography matrix. It selectively purifies biomolecules based on their biological function or chemical structure in a single step. The technique involves equilibrating a column packed with a ligand-coupled matrix, adding a sample for the target substance to adsorb, washing away non-adsorbed components, and then eluting the target component bound to the ligand. Affinity chromatography offers high selectivity and resolution to effectively extract and purify target components from complex biological mixtures.

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    Affinity Chromatography: Techniques in Biochemistry

    Uploaded by

    Sadia Din
    75 views13 pages
    Affinity chromatography is a separation and purification method that exploits the biological affinity between a protein and ligand coupled to a chromatography matrix. It selectively purifies biomolecules based on their biological function or chemical structure in a single step. The technique involves equilibrating a column packed with a ligand-coupled matrix, adding a sample for the target substance to adsorb, washing away non-adsorbed components, and then eluting the target component bound to the ligand. Affinity chromatography offers high selectivity and resolution to effectively extract and purify target components from complex biological mixtures.

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    affinity chromatography

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    Affinity chromatography is a separation and purification method that exploits the biological affinity between a protein and ligand coupled to a chromatography matrix. It selectively purifies biomolecules based on their biological function or chemical structure in a single step. The technique involves equilibrating a column packed with a ligand-coupled matrix, adding a sample for the target substance to adsorb, washing away non-adsorbed components, and then eluting the target component bound to the ligand. Affinity chromatography offers high selectivity and resolution to effectively extract and purify target components from complex biological mixtures.

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    75 views13 pages

    Affinity Chromatography: Techniques in Biochemistry

    Uploaded by

    Sadia Din
    Affinity chromatography is a separation and purification method that exploits the biological affinity between a protein and ligand coupled to a chromatography matrix. It selectively purifies biomolecules based on their biological function or chemical structure in a single step. The technique involves equilibrating a column packed with a ligand-coupled matrix, adding a sample for the target substance to adsorb, washing away non-adsorbed components, and then eluting the target component bound to the ligand. Affinity chromatography offers high selectivity and resolution to effectively extract and purify target components from complex biological mixtures.

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    © All Rights Reserved
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    Download as pptx, pdf, or txt
    of 13

    AFFINITY

    CHROMATOGRAPHY
    T E C H N I Q U E S I N B I O C H E M I S T RY
    AFFNITY CHRMOATOGRAPGHY

    • Biological substances, such as enzymes and substrates or


    antigens and antibodies, have the property of selectively
    recognizing each other to form complexes.
    • Affinity chromatography separates proteins on the basis of a
    reversible interaction between a protein and a specific ligand
    coupled to a chromatography matrix.
    • Affinity chromatography is a separation and purification
    method that exploits this biological affinity.
    SPECIFICITY

    • it is the only technique that enables the purification of a


    biomolecule on the basis of its biological function or individual
    chemical structure.
    • In a single step, affinity purification can offer immense time-
    saving over less selective multistep procedures.
    • The concentrating effect enables large volumes to be processed.
    • Target molecules can be purified from complex biological
    mixtures.
    • small amounts of biological material can be purified from high
    levels of contaminating substances.
    PRINCIPLE

    • In principle, affinity chromatography can effectively extract a


    target component from a complex matrix that contains
    multiple components just using simple operations.

    • The technique offers high selectivity, hence high resolution,


    and usually high capacity for the protein(s) of interest.
    PROCEDURE FOR AFFINITY
    CHROMATOGRAPHY
    • The procedure comprises the following four steps:
    1. Column equilibration:
    The column is filled with a packing onto which a suitable ligand is immobilized.
    The column is then equilibrated with the solvent that will be used for sample
    adsorption.
    2. Sample addition and adsorption of target substance:
    The sample is introduced into the column and the target component adsorbed
    onto the packing.
    3. Column washing:
    Washing is conducted to eliminate components not adsorbed onto the packing
    from the column.
    4. Elution of target components:
    A solvent is introduced into the column to elute the target component adsorbed on
    the packing to recover the target component. The procedure then reverts to step.
    LIGAND SELECTION AND
    IMMOBILIZATION ON THE PACKING
    • The selection of the ligand and packing matrix and method to
    immobilize the ligand must be carefully considered before
    conducting actual affinity chromatography.
    • Selecting the Matrix Commonly used matrices are agarose and
    hydrophilic synthesized macromolecules (polyvinyl alcohol,
    polyacrylate, etc.) beads.
    ELUTION

    • pH elution
    A change in pH alters the degree of ionization of charged groups
    on the ligand and/or the bound protein. This change may affect
    the binding sites directly, reducing their affinity, or cause
    indirect changes in affinity by alterations in conformation.
    • Ionic strength elution
    The exact mechanism for elution by changes in ionic strength
    will depend upon the specific interaction between the ligand and
    target protein. This is a mild elution using a buffer with
    increased ionic strength (usually NaCl), applied as a linear
    gradient or in steps.
    • Competitive elution
    Selective eluents are often used to separate substances on a
    group specific medium or when the binding affinity of the
    ligand/target protein interaction is relatively high. The eluting
    agent competes either for binding to the target protein or for
    binding to the ligand.
    • Reduced polarity of eluent
    Conditions are used to lower the polarity of the eluent promote
    elution without inactivating the eluted substances. Dioxane (up
    to 10%) or ethylene glycol (up to 50%) are typical of this type of
    eluent.
    THE END

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