[molecular biology work] ¶ Gel filtration chromatography

[Seung Jae Jeong]

¶ Gel filtration chromatography

 

–          150Ø(293 cell) 3장에 construct transfection. 각 dish당 14ug DNA transfection.

–          Overnight culture(20~24시간)

–          cold 1X PBS 10ml로 1회 washing.

–          hypotonic solution :10mM Hepes pH7.6, 1.5mM MgCl2, 10mM NaF, 10mM KCl, 0.5mM EGTA, 1mM PMSF, 12mM b-glycerophosphate, 1mM sodium vanadate, 1mM DTT – 3ml로 transfected 293 cell harvest.

–          30 min. on ice

–          3ml syringe로 7회 suction.

–          4°C, 33,000 g에서 20min.간 centrifuge

–          150mM KCl hypotonic sol.과 sample을 1:1로 mix.

–          10mg/ml이 되도록 농축 그리고 0.22um filter로 fitering

–          80mM KCl hypotonic sol.을 이용하여 gel filtration 실시.

: sample 500ulfh injection, superdex HR200이용, flow rate 0.5ml/min, fration size 500ul.

 

 

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