Preparation of cell lysates from E. coli by enzymatic lysis
蚂蚁淘
/发布时间:1545759651 /浏览量:202
PreparationofcelllysatesfromE.colibyenzymaticlysisMaterialsChemicals 50-200mMNaCl* 5%glycerol(v/v) 1mMDTT 1mMPMSF
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Lysisbufferlysosyme
StocksolutionsTheNaClconcentrationusedinthelysisbufferdependsfullyontheapplication.IncaseofaffinitychromatographyonaNi-columntheNaClconcentrationisusually200mMbutwhenthefirstpurificationstepisionexchangechromatographynosaltshouldbeadded.
Procedure1mg/mlDNaseandinwater 100mMPMSF(phenylmethylsulfonylfluoride)inisopropanol 1MMgCl2 1. ResUSPendthecellsinchilledlysisbufferinaratioof1gcellwetweightto1mllysisbuffer. 2. Addlysosymetoafinalconcentrationof300µg/mlandincubatethecellsuspensionat4°Cfor4h. 3. Add5µlMgCl2(1M)and1µlDNasesolution(1mg/ml)permlofcellsuspensionandincubatethesolutionat4°Cfor30min. 4. Removecelldebrisbyultracentrifugationat4°Cfor30minat45000rpmusinga45Tirotor(Beckman).
