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PreparationofcelllysatesfromyeastusingglassbeadsvortexingEquipmentandreagentsLysisbuffer| 50mMTris-HClpH8.0 1%DMSO 50-200mMNaCl* 1mMEDTA 1mMPMSF 1µg/mlleupeptin 1µg/mlpepstatinA | * | TheNaClconcentrationusedinthelysisbufferdependsfullyontheapplication.IncaseofaffinitychromatographyonaNi-columntheNaClconcentrationisusually200mMbutwhenthefirstpurificationstepisionexchangechromatographynosaltshouldbeadded. |
Stocksolutions| 100mMPMSF(phenylmethylsulfonylfluoride)inisopropanol | | 1mg/mlleupeptininwater | | 1mg/mlpepstatinAinmethanol |
EquipmentPreparationofglassbeads| 1. | Soaktheglassbeadsfor16hinconcentratedHCl. | | 2. | Rinsethorouhglyindistilledwater. | | 3. | Bakethemfor16hatabove150°C. | | 4. | Chilltheglassbeadsat4°Coronicebeforeuse. |
ProcedureThisprocedureissuitableforsmallsamples(upto15ml).Forlargersamples,specializedapparatushavebeendeveloped,suchasBiospecProductsBeadBeater,whichallowscellsUSPensionsupto200mltobesubjectedtoagitationatonce.| 1. | PreparetheglassbeadsbywashingtheminconcentratedHCl,followedbyextensiverinsing(checkthatthenpHisneutral)anddrying.Thedriedbeadsshouldbechilledbeforeuse. | | 2. | Resuspendthecellsinanequalamountofchilledlysisbufferandplacethesuspensioninasturdytube(preferablynotglass).- AddthePMSF(10µlPMSF(100mM)permlofcelsuspension)atthispoint.
| | 3. | Add1-3gofchilledglassbeadspergramofcellwetweight. | | 4. | Vortex3-5timesfor1minute,eachtimekeepingthecellsonicefor1minutebetweenvortexings.usethehighestsettingofthevortexmixer. | | 5. | Removetheglassbeads. | | 6. | Removecelldebrisbyultracentrifugationat4°Cfor30minat45000rpmusinga45Tirotor(Beckman). |
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