Next Experiment: Western Blot of Myoglobin

1. Next Experiment: Western Blot of Myoglobin Brief Overview Intro to Immunology

2. 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

3. 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

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

5. 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

6. 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

7. 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?

10. In our experiment, the immobilized metal is Zn+2

11. Mb has 12 inherent his with high Zn+2 affinity

16. Antibody binding is highly specific How is this specificity mediated? Complementarity: receptor/ligand binding is essentially pattern recognition between two complementary surfaces.

17. Heavy chains Light chains Ag: Angiotensin II

18. Ag: HIV protease fragment

23. Stryer Fig. 4.36 Western blot.

26. Antibodies make great antigens!

28. One antigen, many epitopes, many Ab classes One antigen, one epitope, one Ab class

40. Enzyme Linked Immunosorbent Assay (ELISA)

43. Immunotherapeutics