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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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slide1

Next Experiment:

Western Blot of Myoglobin

Brief Overview

Intro to Immunology

slide2

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.

Page 244

slide3

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

slide5

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
slide6

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
slide7

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?

slide11

Mb has 12 inherent his

with high Zn+2 affinity

antibody binding is highly specific
Antibody binding is highly specific

How is this specificity mediated?

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

slide17

Heavy

chains

Light

chains

Ag: Angiotensin II

slide28

One antigen,

many epitopes,

many Ab classes

One antigen,

one epitope,

one Ab class

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