Preparation of Conventional Actin from Skeletal Muscle相关交流-蚂蚁淘在线

Preparation of Conventional Actin from Skeletal Muscle

蚂蚁淘 /发布时间:1545758281 /浏览量:50

ModifiedfromSpudich&Watt,1971,JBC246:4866.

1.Mix20mlbufferGwitheachgmofmuscleacetonepowder.Extractwithstirringonicefor30min.Useabigstirbaroroverheadstirrer.BufferG:0.2mMATP,0.5mMDTT,0.2mMCaCl₂,2mMTris/HCl,pH8.0@25°.

	Stock	1l	4l
2mMTris/HCl,pH8@25°	1M	2ml	8ml
0.2mMATP	0.1M	2ml	8ml
0.5mMDTT	dry	77mg	308mg
0.2mMCaCl₂	1M	0.2ml	0.8ml

2.Centrifugeat16,000rpmfor30mininJ-20,SS-34orA600incold.IfdoingasperCapZprep,filterthroughcheeseclothasforacetonepowder.

3.Filtersupernatantthruglasswoollayer-coarse&rapid.

4.ResUSPendpelletinsamevolumeandrepeatsteps2and3.

5.Combinethesupernatants,stirgently,andmakethem50mMinKCl(2.5mlof2MKClper100ml)and2mMMgCl₂,(0.2mlof1MMgCl₂per100ml).

6.Stiratroomtempfor15minthenonicefor15min,whichpolymerizestheactin.

7.Whilestirring,make0.8MinKCl(23.4mlof4MKClper100ml),whichdissociatestropomyosin.Stirslowlyincoldfor15min.

8.Centrifuge2hrsinTi42or45rotorat35,000rpm.Discardsupernatant.Shouldseeclear/translucentgelatinouspellet.RinsepelletoncewithbufferG.

9.Carefullyliftentirepelletoutoftubebyslidingspatulaunderneath.TransfertoDouncehomogenizer.RinseremainderofpelletoutoftubewithbufferG.

10.Suspendpelletsintotalabout3mlbufferGpergmofacetonepowderandhomogenizewithDounce.Shouldbeveryviscous,evendifficulttotransfertodialysistubing.

11.Dialyseinthinnestdialysistubingpracticalfor2-3days.ChangethebufferGeverydayorhalfday.

12.SpininTi42or45for2hrsat35,000rpm.Carefullyremovetop2/3ofsupt.Thisis"conventionalactin."Bottom1/3ofdifferenttubescanbecombinedandre-spun,butwouldnottrytomakeG150actinfromthis.

13.MeasureconcentrationbyA₂₉₀.Extinctioncoefficientis0.63ml/mg/cmor26,600M^-1cm^-1.

14.ForgelfiltrationonSephadexG150,diluteto5mg/mlwithbufferG.Samplesizeshouldbe<=5%ofcolumnvolume.Collect4mlfractions.Columnprofileshouldbesmallpeakatvoidvolume,largepeakintheback.Thelargepeakshouldbeskewedtowardthefront.ThefrontofthispeakiscontaminatedwithCapZ,soavoidit.

15.Poolfractionsandmeasureconcentration.Storebyadding2mgsucrosepermgactin.Quick-freezeandlyophilize.

16.Touse,dissolveanddialyzeovernightvsbufferG.Inahurry,youcanusethedissolvedmaterialwithoutdialysis,butrememberthatitnowhasextraATPfromthelyophilizedbufferG,sotheabsorbanceistoohigh.

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