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Next Experiment: Western Blot of Myoglobin. Brief Overview Intro to Immunology. Figure 8-39b Representations of the X-ray structure of sperm whale myoglobin. ( b ) A diagram in which the protein is represented by its computer-generated C a backbone. Page 244.

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Next Experiment: Western Blot of Myoglobin

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Next Experiment:

Western Blot of Myoglobin

Brief Overview

Intro to Immunology


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Figure 8-39bRepresentations of the X-ray structure of sperm whale myoglobin. (b)A diagram in which the protein is represented by its computer-generated Ca backbone.

Page 244


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Figure 8-39cRepresentations of the X-ray structure of sperm whale myoglobin. (c)A computer-generated cartoon drawing in an orientation similar to that of Part b.

Page 244

Mb structure

Protein Workshop


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http://www.merck.de/servlet/PB/menu/1141240/index.html


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  • Affinity Chromatography

  • Affinity chromatography can be used to purify proteins that have a high specific affinity to some chemical group

  • For example, concanavalin A is a sugar-binding protein with a high affinity for glucose (and mannose)

  • A column with glucose residues attached will retain concanavalin A, which can then be later released by adding a solution of free glucose

  • This technique is not always be applicable because the specific high-affinity groups are not always available

  • For certain categories of proteins such as transcription factors, the high affinity group can be a specific sequence of DNA, which is easily prepared


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    • Affinity Chromatography

  • Affinity chromatography can be used to purify proteins that have a high specific affinity to some chemical group

    • For example, concanavalin A is a sugar-binding protein with a high affinity for glucose (and mannose)

    • A column with glucose residues attached will retain concanavalin A, which can then be later released by adding a solution of free glucose

    • This technique is not always be applicable because the specific high-affinity groups are not always available

    • For certain categories of proteins such as transcription factors, the high affinity group can be a specific sequence of DNA, which is easily prepared


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    Immobilized Metal Ion Affinity Chromatography (IMAC)

    Immobilized Metal Ion Affinity Chromatography (IMAC) with Ni2+ is the standard method for purifying proteins with a lot of surface histidine or for engineered His-tagged proteins.

    The (His)6-tag has become the most used affinity tag due to its small size, strong metal ion binding and ability to bind also under denaturing conditions.

    How could you create a his tag?


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    In our experiment, the immobilized metal is Zn+2


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    Mb has 12 inherent his

    with high Zn+2 affinity


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    Antibody binding is highly specific

    How is this specificity mediated?

    Complementarity: receptor/ligand binding is essentially pattern recognition between two complementary surfaces.


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    Heavy

    chains

    Light

    chains

    Ag: Angiotensin II


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    Ag: HIV protease fragment


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    Stryer Fig. 4.36 Western blot.


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    Antibodies make great

    antigens!


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    One antigen,

    many epitopes,

    many Ab classes

    One antigen,

    one epitope,

    one Ab class


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    Enzyme Linked Immunosorbent Assay (ELISA)


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    Immunotherapeutics


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