Next Experiment:

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Next Experiment Western Blot of Myoglobin
Brief Overview Intro to Immunology
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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 Ca backbone.
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Figure 8-39c Representations 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 Zn2
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Mb has 12 inherent his with high Zn2 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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