常见重组蛋白纯化方法

(Chromatography)@263.net1112346XHispHGST10-20mMMBPAIgGpHFlagpeptideFlag,M1,M2pHEDTAPoly-ArgSPPoly-CysDTTPoly-PheEGTAGST2His-TagIMAC21IMAC(ImmobilizedMetal-ionaffinitychromatography)Porathetal.1975IDA1987Smithetal.IDA-sephadexG-251986Ni2+-IDA-sephadexG-251987Hochulietal.Ni2+-NTA1988IMACIMAC1986Porathetal.Fe3+-IDA-sephadexG-25Ga3+-IDAIMACIMACIDA33NTA42CM-Asp42TED51FFIDA40umol/ml10mg/ml70mg/mlNTAFFNTA20umol/ml5-10mg/ml2.2IMAC2.2.12.2.2IDANTANTAIDAIDANTANTA.2.2.2IMACNTA1-2M8M6M1-2mMDTT2.2.3pHTris-HClpHpH0.1-1M00.1-0.5%triton2.2.40.5-1%NTA1-2mM147000-8000g10(-70)21-210mlpH7.450mM0.5MNaCl0.5mg/ml1mMPMSF1mMMgCl21.7units/mlBenzonase,1mMPMSF1.7units/mlBenzonase45pH7-80.5MNaOH.10mg/ml10.320,,2,pH,7-8,0.5MNaOH.50-500,,,1,,800bar.4412000g10450000g3052M0.12ml20mM10ml0.45m21pH7.450mM0.5MNaCl20mM2pH7.450mM0.5MNaCl500mM31mlFFNTAFF10ml10ml0.5ml/min2ml/415ml1-2ml/min2ml/55ml1-2ml/min2ml/65ml20%750mM100mM300mM500mM58BCA31pH212NTA3pHpH3500mM500mM6M40.5MpH50.5%Triton1-2mMNTA1)47000-8000g10(-70)1-210mlpH8.550mMTris-HCl2mMEDTA100mMNaCl0.5%TritonX-1001mg/ml45)20,,2,pH,8.5,1MTris.50-500,,,1,,800bar.)412000g10450000g30)4MpH7.450mM0.5MNaCl8M20mM0.45m41pH7.450mM0.5MNaCl8M20mM2pH7.450mM0.5MNaCl8M500mM31mlFFNTAFF10ml10ml0.5ml/min2ml/415ml1-2ml/min2ml/55ml1-2ml/min2ml/65ml20%750mM100mM300mM500mM58BCA91pH2-5mM212NTA3pHpH3500mM500mM6M40.5MpH50.5%Triton1-2mMNTAGST547000-8000g10(-7061-210mlpH7.450mM0.5MNaCl0.5mg/ml1mMPMSF1mMMgCl21.7units/mlBenzonase,1mMPMSF1.7units/mlBenzonase45pH7-80.5MNaOH.10mg/ml10.720,,2,pH,7-8,0.5MNaOH.50-500,,,1,,800bar.8412000g10450000g30950.45m)150mMTris-HClpH8.30.1MTris50ml0.1M20ml0.9gNaCl100ml250mMTris-HClpH8.30.1MTris50ml0.614g0.1MpH8.3100ml()31mlGSTFF10ml10ml0.5ml/min2ml/410ml1-2ml/min2ml/55ml1-2ml/min2ml/65ml20%7BCA1pH21GSTGSTGSTGST1-5mMDTT2GST103pHpH4pH6M0.5MNaOH5-105GSTGST26KD0.5%Triton1-2mM