Expanded Bed ID:M9506025 Student : 郭育秀 2007/01/08

Outline Introduction Experiment procedure Conclusion 1.Expanded bed 2.Immobilized-Metal Affinity Chromatography (IMAC) Experiment procedure Conclusion

Expanded Bed Adsorption principle 1.Based on controlled fluidization of porous adsorbent resin. 2.Increasing the space between the adsorbent particles. 3.The beads of EBA are variable. (the larger particles populate the lower portion of fluidized bed while the smaller particles populate the upper portion)

Expanded Bed Adsorption principle Fig1. Comparison between a) a packed bed and b) a stable,expanded bed.

Expanded Bed Adsorption principle Fig2. Theory of expanded bed adsorption

Immobilized-Metal Affinity Chromatography (IMAC) The technique is based on difference in the affinity of protein for metal ions bound to a metal-chelating substance which is immobilzed on a chromatogrphic support.

Fig.3 The mechanism of IMAC. E.K.M. Ueda et al./j.chromatogr.A 988(2003)1-23

IMAC Fig.4. Structures of two commonly used chelating resins charged with Ni (II) ions : Ni-IDA and Ni (II)-NTA E.K.M. Ueda et al./j.chromatogr.A 988(2003)1-23

IMAC E.K.M. Ueda et al./j.chromatogr.A 988(2003)1-23

IMAC V. Gaberc-Porekar, V. MenartrJ. Biochem. Biophys. Methods 49(2001)335–360

Adding biomass to the column Experiment procedure Removing ethanol from expanded bed Copper priming and equilibrating with buffer. Adding biomass to the column Elution the column Cleaning the expanded-bed system 1.Sanitizing the expanded bed with NaOH 2.Neutralizing the expanded-bed system 3.Storing the expanded-bed system in ethanol.

Experiment procedure A: collapsed state. B: copper loading C: washing off unbound copper. D: starting to load biomass. E: middle of loading biomass. F: washing off cells and cell debris. G: washing with 40mM imidazole H: elution with 200mM I: washing with EDTA J: washing with NaOH Nature protocols/2006/V1.N3 p1213~p1222

Conclusion Advantage of IMAC: 1.Only need single step to purify. 2.The protein loading capacity is high. 3.The metal ion can be easily to remove. 4.Useful for concentrating dilute protein solution. 5.IMAC resin can be reused for hundreds of times.

Conclusion Disadvantage of IMAC: 1.Metal toxicity 2.Removal of histidine tags 3.During oxidative or catalytic conditions, the metal ion will tend to undergo redox reaction.

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