Preparation of cell lysates from E. coli by sonication
来自 : 蚂蚁淘
PreparationofcelllysatesfromE.colibysonicationMaterialsLysisbuffer| 50mMTris-HClpH7.5 50-200mMNaCl* 5mMDTT 1mMPMSF | * | TheNaClconcentrationusedinthelysisbufferdependsfullyontheapplication.IncaseofaffinitychromatographyonaNi-columntheNaClconcentrationisusually200mMbutwhenthefirstpurificationstepisionexchangechromatographynosaltshouldbeadded. |
Stocksolution| 100mMPMSF(phenylmethylsulfonylfluoride)inisopropanol |
Procedure| 1. | ResUSPendthecellsinchilledlysisbuffer.Normallyratiosofcellwetweighttobuffervolumeof1:1to1:4areused. | | 2. | Coolthecellsuspensiononicefor10min.- Add10µlPMSF(100mM)permlofcelsuspensionafterthisstep.
| | 3. | Sonicatethecellsuspensionwith10shortburstof10secfollowedbyintervalsof30secforcooling.- Keepthesuspensionatalltimesonice.
- Avoidfoaming.
- Don’tgoawaywhilethesonicatorisinoperation.ItispossIBLethatthebeakerbreaksorturnsinthemeltingice.
| | 4. | Removecelldebrisbyultracentrifugationat4°Cfor30minat45000rpmusinga45Tirotor(Beckman). |
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