Introductions: The kitprovidesan optimizedreagentand buffer system for efficientextraction of biologically active and soluble total protein from yeast cells. The kit contains EDTA、CaCl2, DTT, protease inhibitors, as well as a proprietaryreagent to break cell wall. Native and/or recombinant proteins extracted are preserved with biological activity integrities and are suitable for many downstream applicationsincludingSDS-PAGE,Westernblot,enzymeanalysis,2Dgelelectrophoresis.Thekitis sufficientfor50x50mgwetyeastcells.
Compositions:
Isoosmotic Buffer
75ml
SnailaseBuffer
250ul
Hypoosmotic Buffer
25ml
PMSF Solution
250ul
Storage: Transportationatroomtemperature.Uponreceipt,storeIsoosmoticBuffer,HypoosmoticBuffer at4oC. Store the Snailase buffer and PMSF Buffer at-20oC.
Procedures: 1.Cultivate the yeast strain in suitable medium at 28oC to 30oC until OD600 of yeast cells ensity reaches ~1.0orso. 2.Centrifuge at 8000rpm for one minute. Discard supernatant and keep yeast paste. Record the weightofwetpaste. 3.Add500ulIsoosmoticbuffer,5ulSnailasebufferand1ulmercaptoethanol(notprovidedinthekit)per50mgwetyeastpaste.
Pipette themixedsolution upanddown tofullyre- suspendyeastcells. 4.Incubate at30oC forone hour. Occasionallyinvert the tubeseveral times. 5.Centrifuge at5000rpmforoneminute,discardsupernatantandkeepprecipitates.
6.Washtheprecipitateswith500ulIsoosmoticbuffer.Thencentrifugeat5000rpmforoneminutes.
Discardsupernatantandkeepprotoplasmic precipitates. 7.Repeatstep6oncemore.
8.Add500ulHypoosmoticbufferand5ulPMSFsolution.Vortexandkeepsolutionat-20oCfor30minutes.
Then thawatroom temperature.Repeatfreeze-thawoperation oncemore.
9.Centrifugetheabovelysedsolutionat5000rpmfor5minutes.Transfersupernatantforwestern blot,SDS- PAGE
,and/orimmunoprecipitation experiments orstore at- 20oC till urtheruse
Notes: 1.For50mgwetyeast,add500ulIsoosmoticbuffer,5ulSnailasebuffer.Toomuchyeastcellsused will lead toincompletelysis ofcellwall. 2.Theabovelysedsolutionmustbestoredcold at2-8oC for thefurtherexperiments
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