Isolation of newly synthesised myosin filaments from skeletal...
来自 : 蚂蚁淘
IsolationofActinandMyosinFilamentsLEVELIII
Materials
- RelaxingSolution
- 0.1MKCl
- 0.001MMgCl
- 5mMATP
- 0.016MNaHPO
- NaHPO
- AdjustpHto7.3
0.05MSodiumphosphatebuffer,pH7.00.001MEDTA(Ethylenediaminetetraaceticacid)BlenderPreparativecentrifugeMaterialsforTEMfixation,embeddingandobservationProcedure7
- Obtainfreshchickengizzards8anddissectthelateralmusclesfreefromallattachments.Placethemusclesoncrushediceandthengrindtheminastandardworm-drivemeatgrinder.Smallsamplescanbepushedthroughahandpress,ifdesired.
- Weighthetissueandaddanequalvolumeofcold0.05MSodiumphosphatebuffer,pH7.0with0.001MEDTA.Blendthismixinastandardblenderatlowspeedandpourtheslurryintoalargebeaker.
- Uponsettling,themusclefragmentswillsettleontopoftheunderlyingconnectivetissue.Separatethetwobydecanting,andconcentratebylowspeedcentrifugation.
- SUSPendaliquotsofblendedmuscleintwovolumesofrelaxingsolutionandhomogenizeinablenderathighspeedfor30seconds.
- Centrifugethehomogenateat500xgtoremovemembraneousorganellesandwholecells.Themyofilamentsarepreferentiallylocalizedinthemiddle,clearsolutionofthecentrifugetube.
- Collectthemiddlelayerandrecentrifugeat40,000xgtopelletthemyofilaments.Washandgentlyresuspend.Thiswillridthepreparationofsolubleproteins.
- PrepareasmallsampleofthemiddlelayerfromStep5forEMobservation,followingtheprocedureoutlinedabovefortubulin(Exercise9.7).
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