1. http://www.youtube.com/watch?v=dd7Q7vhN B-I&feature=related

2. Metabolism0.1 mM, 1x 10 8 Ribosomes 10  1x 10 7 Kinases Cyclins 1  1x 10 6 0.1  1x 10 5 Transcription factors 10 nM, 1x 10 4 Synaptic Markers 0.1 nM, 1x 10 3 Cytoskelatal Proteins mM, 1x 10 9 copies/cell Are We Ready for Mammalian Proteomics ?

4. Protein Purification Grow cells in media (vector+tag) Centrifuge, Collect the pellet Lyse the cells (appropriate buffer) Purification Strategy SDS PAGE, Assay Solubility Aggregation Recombination Characterization Mass Spectroscopy X-ray Crystallography Functional Assay

5. OUTLINE Chromatography techniques Affinity Chromatography (AC) Hydrophobic Interaction Chromatography (HIC) Ion Exchange Chromatography (IEC) Gel Filtration (GF) Capillary electrochromatography (CEC) Other New Strategies for Protein Purification Solubility, Aggregation and Re-folding of Proteins Invented by a Russian botanist named Mikhail Tswett in 1903. He separated plant pigments using glass columns packed with calcium carbonate.

6. OUTLINE Chromatography techniques Affinity Chromatography (AC) Hydrophobic Interaction Chromatography (HIC) Ion Exchange Chromatography (IEC) Gel Filtration (GF) Capillary electrochromatography (CEC) Other New Strategies for Protein Purification Solubility, Aggregation and Re-folding of Proteins Invented by a Russian botanist named Mikhail Tswett in 1903. He separated plant pigments using glass columns packed with calcium carbonate.

7. Affinity Chromatography Surface bound with Epoxy, aldehyde or aryl ester groups Metal Interaction Chromatography Surface bound with Iminodiacetic acid + Ni 2+ /Zn 2+ /Co 2+ Affinity Chromatography (Christian G. Huber, Biopolymer Chromatography, Encylcopedia in analytical chemistry, 2000)

8. Metal Interaction Chromatography (AC) Points to Note: 1.Avoid chelating agents 2.Increasing incubation time 3.Slow gradient elution (www.qiagen.com)

9. Biopolymer (phenyl agarose - Binding Surface) Driving force for hydrophobic adsorption Water molecules surround the analyte and the binding surface. When a hydrophobic region of a biopolymer binds to the surface of a mildly hydrophobic stationary phase, hydrophilic water molecules are effectively released from the surrounding hydrophobic areas causing a thermodynamically favorable change in entropy. Temperature plays a strong role Ammonium sulfate, by virtue of its good salting-out properties and high solubility in water is used as an eluting buffer Hydrophobic Interaction Chromatography Hydrophobic region (Christian G. Huber, Biopolymer Chromatography, Encylcopedia in analytical chemistry, 2000)

10. Fractogel matrix is a methacrylate resin upon which polyelectrolyte Chains (or tentacles) have been grafted. (Novagen) Ion Exchange Chromatography Globular Protein Deformation due to interaction with conventional ion exchanger Maintenance of conformation while interacting with tentacle ion exchanger (www.novagen.com)

11. Gel Filtration (http://lsvl.la.asu.edu/resources/mamajis/chromatography/chromatography.html)

12. Immuno Affinity Chromatography (http://www.cellmigration.org/resource/discovery/discovery_proteomics_approaches.html)

13. Edman Sequencing

15. Carboxypeptidase

16. Carboxypeptidase Mechanism