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来自 : 蚂蚁淘
LysosomeIsolationinIsotonicSucroseLEVELIMATERIALS
- Ratliver
- Physiologicalsaline(0.85%w/vNaCl)
- 0.25Msucrosein10mMTris-HCL,pH7.4
- Brendlerteflonhomogenizer
- Refrigeratedpreparativecentrifuge
- 0.08MCaCl
in0.25Msucroseplus10mMTris-HCl - 150mMKClin10mMTris-HClBuffer,pH7.4
- Phasecontrastmicroscope,slides,coverslips
PROCEDURE4
- Decapitateandexsanguinatearatthathasbeenstarvedforatleast24hourspriortothelab.5
Fillasyringewithsalineandgentlyperfusetheliverbyforcingthesalinethroughthehepaticportalvein,andthroughtheliver.
- Removetheliver,placeitinapreweighedbeakerandweighthebeakerandliver.Calculatetheweightoftheliver.
- Preparea10%(w/v)homogenateorbrei.Foreachgramofliver,add9.0mlof0.25Msucrosein10mMTris-HCL,pH7.4.tothebeaker.
- Gentlychoptheliverinthesucroseandtransferthechoppedlivertoateflonhomogenizer.6
Gentlyhomogenizetheliverwhilekeepingitchilled.
- Centrifugethebreiat12,000xgfor10minutesat4°C.Decantthesupernatantintoachilledbeakeranddiscardthepellet.
- Add0.08MCaCltothesupernatanttoyieldafinalconcentrationof8mM(use1mlofCaClper9mlofsupernatant).Stirgentlyandrecentrifugeat25,000xgfor15minutesat4°C
- Carefullyremoveanddiscardthesupernatant.7
- ResUSPendthepellet(containingthelysosomes)in30mlof150mMKClin10mMTris-HClBuffer,pH7.4.
- Re-sedimentthelysosomesbyafinalcentrifugationat25,000xgfor15minutesat4°C.
- RemoveasmallportionofthepelletforExercise7.2.Resuspendtheremainderofthepelletin30mlof150mMKCl/10mMTris-HClBuffer.ThissuspensionisthelysosomefractionforExercise7.3.
- Prepareawetmountoftheresuspendedlysosomepelletandobservewithaphasecontrastmicroscopeat100X.Drawanystructuresobserved.
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