Purification of recombinant T7 RNA polymerase

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Purification of recombinant T7 RNA polymerase 생유기화학실험 2011.11.16 실험조교: 김지은, 김효선

Concentrated ammonium sulfate 1) Cell lysis and protein fractionation Concentrated ammonium sulfate Cell lysis supernatant protein pellet H2O + ammonium sulfate P P ammonium sulfate H2O < Salting-out effect>

1) Cell lysis and protein fractionation (cont’d) H2O + ammonium sulfate Lysis Buffer Dialysis (O/N) membrane

2) Purification with Column Chromatography Dialysis salt Sephadex resin Lysate 1) Washing buffer 2) Elution buffer

－－－－－－－－－－ * Ion-exchange column chromatography - - SP Sephadex C-50 are strong cation exchangers. Sephadex resin － + protein - + － + － + －－－ Elution w/NaCl － resin - －－－ SP: Sulfopropyl group(구조?) + + +

2) Concentration and Quantitation of the purified protein. Ultra-filtration :concentration Bradford assay (quantitation) 12% SDS-PAGE (Eluant, loading sample, washed fraction…) Dialysis (O/N) for buffer exchange Store protein sample @ -20 oC for further experiment

HOME WORK 결과 리포트 : 각각의 시약과 buffer의 역할 및 작용 원리 (Chromatography resin도 포함) 예비 리포트: in vitro Transcription의 방법 과 원리 (T7 RNA polymerase system)