Preparation of cell lysates from yeast by enzymatic digestion of the cell wall
来自 : 蚂蚁淘
PreparationofcelllysatesfromyeastbyenzymaticdigestionofthecellwallMaterialsChemicalsLyticase[orZymolase100T(ICN,USA)] |
Brij58 |
Workingbuffer50mMTris-HClpH8.0 10%sorbitol Stocksolutions 100mMPMSF(phenylmethylsulfonylfluoride)inisopropanol | 1mg/mlleupeptininwater | 1mg/mlpepstatinAinmethanol | 100mMspermidine(pH7.8) | 100mMEDTA | 1MMgCl2. | 4MKCl | Procedure1. | ResUSPendthecellsinanequalamountofchilledworkingbuffer.AdjustthepHofthesuspensionto8.2withsolidTrisbase. | 2. | Freezethesuspensionrapidlyinliquidnitrogeninanultracentrifugetube.(Freezingandthawingisessentialinthisprocedure). | 3. | Thawthefrozensuspensionisthawedrapidlyandplaceitonice.- Add10µlPMSF(100mM)permlofcelsuspensionatthispoint.
| 4. | Addthefollowingsolutionstothecellsuspension:4MKCltoaconcentrationof1M,100mMspermidineto5mM,andlyticaseto2000U/ml.- Insteadoflyticase,Zymolase100Tcouldbeusedatafinalconcentrationof0.5mg/ml.
| 5. | Mixgentlyandincubatethesuspensionfor30minonice,withoccasionalswirling.Usuallyover90%ofthecellsareconvertedtospheroplasts. | 6. | Adddifferentproteaseinhibitors:2µl/mlEDTA(0.5M),1µl/mlPMSF(100mM),1µl/mlleupeptin(1mg/ml),and1µl/mlpepstatinA(1mg/ml)tothesuspension. | 7. | AddBrij58toaconcentrationof0.1%andheatshockthespheroplastsfor3minat37°C. | 8. | Removecelldebrisbycentrifugationat4°Cfor30minat45000rpmusinga45Tirotor(Beckman). |
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