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

2. 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>

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

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

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

6. 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 -20 oC for further experiment

7. 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)