Chapter 7 ???? Chromatography

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Chapter 7????Chromatography
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????(????)

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  ?????????(???????),??????(??-??-??-??),????????

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• ????
• ??Langmuir????
• Kd---????
• q?c---????????????

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• ????(tR)?????(VR)
• ????????????????,???????????????

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• ????Rt
• ??????????????????????(????????????? Kd0)??????
• ????????????????????????

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• ????
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• ?????,?????????????,?????????

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• ??????????????
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• ??????????
• N---????? tR---????
• W1/2---??? Wb---????
• ??????L---??

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• ??????????????????(????,?????????????????)???????
  ?
• ??????????????
• ??????????????,?????????????????????????,?-OH?-NH
  2

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Adsorption chromatography
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• ????????
• (1)?????????????????,??????????????
• (2)???????????????????

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• ???242243

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• ??????????(?????????????)????????????
• ?????????????(???pH????),??????????,?????????????
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• ?????????????pH?????????,???????????

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(No Transcript)
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• ??????????????????????
• ??P247
• ?????????(?????),????????(????)
• ????????????, ???????

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HPLC
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HPLC Chromatogram
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  ??????????????????????
• ??????????????(????)?????????????

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• ????????(??5µm20 µm )
• Hypersil Spherosil XOB075
• ???C18?C8???,C8???????
• C18??????
• ??
• ??????
• ????????,???????????????

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Source RPC
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Influence of Particle Size on the Separation
effect
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?????????

• ??????
• DEAE-Sephadex A-25,QAE-Sephadex
  A-25,CM-Sephadex C-25,SP-Sephadex C-25
• ??????????
• DEAE-Sephacel Cellex??
• ??????????
• Sepharose?? Sepharose CL-6B, Sepharose Fast
  Flow
• Bio-Gel A ????????????

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• Source ???????
• ???????????????????
• ??????? S??
• ??????? Q??
• MonoBeads ???????(Pharmacia)
• ??????????,???Source???????,???????,?????
• ????Q???P??

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Expanded Bed
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?????????
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Desalting proteins
salts
proteins
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??????(Hydrophobic Interaction Chromatography)

• Hydrophobic interaction chromatography is a
  liquid chromatography technique that separates
  biomolecules according to their hydrophobicity

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Mechanism of HIC
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Na2SO4 1.0 M
??????
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??????

• ?????
• Sephadex G-??
• ???????
• Sephacryl??
• ?????
• Sepharose??
• Superose??

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????(Affinity chromatography)
???????????????
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Competitive elution
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• ??????????,????-??????????????????,??????????
• ???????????
• (1)????
• (2)????????

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  ???????
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• ????????????????

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• ??????????????????
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• ???????????,????pH??,?????????,????????????,??????
  ???,????????
• ????????????,????????,??????????????
• ?????????pH?,????????,?????pH???????(?????)

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• ?????????????,???1020m mol
• ???????,????????????
• ????pH??
• ??????

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Purification of recombinant a-mannosidase from
Pichia pastoris
Technique
Purificationfactor
Y.-F. Liao et al. (1996) J. Biol. Chem. 271, 28348
AIEX
Q Sepharose FF

• 10 times purification
• 82 yield

Phenyl Sepharose HP
HIC
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ÄKTA explorer 100????
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ÄKTA explorer 100????????
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  ???,?????,????,??,????

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• ??????????????????????-??-?(1532)
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• ????(????)
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(No Transcript)
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G Protein Receptor Kinase Purification
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Rec a-Mannosidase Purification from Pichia
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DNA Binding Protein Purification
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• ????1 P281
• ?????????????????????????????
• ????????????????????
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• ??HIC??????
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