细胞分离

...Cell Lysates from Laboratory Escherichia coli Strains and...

PreparationofcelllysatesfromE.coliusingaFrenchPressMaterialsLysisbuffer

50mMTris-HClpH7.5

50-200mMNaCl*

1mMMgCl2

5mMDTT

1mMPMSF

*
TheNaClconcentrationusedinthelysisbufferdependsfullyontheapplication.IncaseofaffinitychromatographyonaNi-columntheNaClconcentrationisusually200mMbutwhenthefirstpurificationstepisionexchangechromatographynosaltshouldbeadded.
Stocksolutions
1mg/mlDNaseinwater.
100mMPMSFinisopropanol.
Equipment
FrenchPresscell(40ml)
Procedure
1.PrechilltheFrenchPresscellat4°C.
2.ResUSPendthecellsinchilledlysisbuffer.Normallyratiosofcellwetweighttobuffervolumeof1:1to1:4areused.Add1µlDNasesolution(1mg/ml)permlofcellsuspensiontoavoidviscosityproblems.
  • Add10µlPMSF(100mM)permlofcelsuspensionatthispoint.
3.ApplythesampletotheFrenchpressurecellandbringthecellunderthedesiredpressure(7000to10,000psi).
4.Whilemaintainingthepressure,adjusttheoutletflowratetoaboutonedropeverysecond.Collectthecelllysateinaflaskthatiskeptonice.
5.Repeatsteps3and4.
6.Removecelldebrisbyultracentrifugationat4°Cfor30minat45000rpmusinga45Tirotor(Beckman).

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