http://www.youtube.com/watch?v=dd7Q7vhNB-I&feature=related

Metabolism 0.1 mM, 1x 108

Ribosomes

10 , mM 1x 107

Kinases

Cyclins

1 , mM 1x 106

0.1 , mM 1x 105

Transcription factors 10 nM, 1x 104

Synaptic Markers 0.1 nM, 1x 103

Cytoskelatal Proteins mM, 1x 109 copies/cell

Are We Ready for Mammalian Proteomics ?

Protein Purification

• Grow cells in media (vector+tag)• Centrifuge, Collect the pellet• Lyse the cells (appropriate buffer)

Purification Strategy

SDS PAGE, Assay

SolubilityAggregation

Recombination

CharacterizationMass Spectroscopy

X-ray CrystallographyFunctional Assay

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.

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.

Affinity ChromatographySurface bound with

Epoxy, aldehyde or aryl ester groups

Metal Interaction ChromatographySurface bound with

Iminodiacetic acid + Ni2+/Zn2+/Co2+

Affinity Chromatography

(Christian G. Huber, Biopolymer Chromatography, Encylcopedia in analytical chemistry, 2000)

Metal Interaction Chromatography (AC)

Points to Note:

1. Avoid chelating agents

2. Increasing incubation time

3. Slow gradient elution

(www.qiagen.com)

Biopolymer (phenyl agarose - Binding Surface)

Driving force for hydrophobic adsorptionWater 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)

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)

Gel Filtration

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

Immuno Affinity Chromatography

(http://www.cellmigration.org/resource/discovery/discovery_proteomics_approaches.html)

Edman Sequencing

Carboxypeptidase

Carboxypeptidase Mechanism