1. Expanded Bed
ID:M
Student : 郭育秀
2007/01/08

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

3. 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)

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

5. Expanded Bed Adsorption principle
Fig2. Theory of expanded bed adsorption

6. 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.

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

8. 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

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

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

11. 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.

12. 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

13. 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.

14. 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.

15. Thanks for your attention!