Expanded Bed

ID:M9506025

Student : 郭育秀2007/01/08

Outline

Introduction 1.Expanded bed

2.Immobilized-Metal Affinity Chromatography

(IMAC)

Experiment procedure Conclusion

Expanded Bed Adsorption principle

Expanded bed 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.

IMAC

Fig.3 The mechanism of IMAC.

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

Fig.4. Structures of two commonly used chelating resins

charged with Ni (II) ions : Ni-IDA and Ni (II)-NTA

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

Removing ethanol from expanded bed

Copper priming and equilibrating with buffer.

Adding biomass to the column

Experiment procedure

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 procedureA: 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

imidazole

I: washing with EDTA

J: washing with NaOHNature 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.

Thanks for your attention!