indlela yokuhlukanisa ama-lysosomes abe yi-peroxisomes nge-centrifugation

Isingeniso kumaLysosome namaPeroxisome

Ama-Lysosome nama-peroxisome angama-organelle amabili ahlukene eseli anemisebenzi ehlukile edlala indima ebalulekile ekusetshenzisweni kwamaseli kanye ne-homeostasis. Ama-Lysosome angama-organelle aboshwe yi-membrane agcwele ama-enzyme okugaya abangela ukubhidliza izinto ezilahliwe zamaseli, izinhlayiya zangaphandle, kanye namagciwane. Ama-Peroxisome, ngakolunye uhlangothi, ahilelekile ekuphenduleni okuhlukahlukene kwe-metabolic, ikakhulukazi lokho okuhlobene nokukhishwa kwe-detox kanye ne-lipid metabolism. Ukuze sitadishe la ma-organelle ngokwehlukana, enye indlela evame ukusetshenziswa i-centrifugation. Kulesi sihloko, sizohlola ukuthi i-centrifugation ingasetshenziswa kanjani ukuhlukanisa ngempumelelo ama-lysosome kuma-peroxisome.

Izimiso Namasu Okusebenza Nge-Centrifugation

I-Centrifugation iyindlela enamandla esetshenziselwa ukuhlukanisa izingxenye ezahlukene zengxube ehlukahlukene ngokusekelwe ngobukhulu bazo, ubuningi, kanye nesimo. Isebenza ngezimiso ze-sedimentation, lapho izinhlayiya ezisindayo zihlala khona ngokushesha ngaphansi kwethonya lamandla e-centrifugal. Ukuhlukanisa ama-lysosome kuma-peroxisome, amasu ahlukene e-centrifugation, njenge-differential centrifugation kanye ne-density gradient centrifugation, angasetshenziswa.

I-Centrifugation Ehlukile: Ukukhipha Ama-Lysosome Nama-Peroxisome

I-centrifugation ehlukile ihilela uchungechunge lwezinyathelo ze-centrifugation ngesivinini esihlukene kanye nobude besikhathi ukuze kuhlukaniswe ama-organelle ngokusekelwe ekubuneni kwawo. Nansi indlela engasetshenziswa ngayo ukukhipha ama-lysosome nama-peroxisome:

Isinyathelo 1: Ukuhlanganiswa kwe-Homogenization

Isinyathelo sokuqala ukuphula amaseli kancane bese ukhulula okuqukethwe kwawo usebenzisa indlela yokwenza i-homogenization. Lokhu kungafezwa ngokufaka i-sonicating noma ukusebenzisa i-homogenizer ekhethekile. Ingxube ephumayo ibizwa ngokuthi i-homogenate.

Isinyathelo 2: I-Centrifugation Esheshayo Esiphansi

I-homogenate ibe isifakwa ku-centrifugation yesivinini esiphansi (cishe i-1,000-2,000 xg) isikhathi esifushane. Lesi sinyathelo sisiza ukuhlukanisa udoti omkhulu wamaseli kanye nengxenye yenuzi kwezinye izingxenye ze-organelles ezikhona ku-homogenate.

Isinyathelo 3: I-Centrifugation Yesivinini Esimaphakathi

I-supernatant etholakala esinyathelweni sokugeleza ngesivinini esiphansi ifakwa ekugelezeni ngesivinini esiphakathi (cishe u-10,000 xg) ukuze ichithe ama-organelle asindayo njenge-lysosomes nama-peroxisomes. Lesi sinyathelo siphumela ku-pellet equkethe la ma-organelles.

Isinyathelo 4: Geza bese Uphinda Usebenzise Ugesi

I-pellet etholwe esinyathelweni sangaphambilini igezwa ngesisombululo esifanelekile esifakwe i-buffer bese iphinda ixhunywe ukuze kutholakale ingxenye ye-lysosome ne-peroxisome ehlanziwe.

I-Centrifugation ye-Density Gradient: Ukuhlanza Ukuhlukaniswa

I-centrifugation ye-density gradient iyindlela ethuthukisiwe ye-centrifugation evumela ukuhlanzwa okwengeziwe kanye nokulungiswa kwama-lysosome nama-peroxisome. Kule ndlela, kwakheka i-density gradient kusetshenziswa isisombululo esakha i-gradient njenge-sucrose noma i-iodixanol. Nansi indlela engasetshenziswa ngayo:

Isinyathelo 1: Ukulungiswa kwe-Density Gradient

I-density gradient medium, njenge-sucrose, ilungiswa ngepayipi le-centrifuge, enobukhulu obuhlukahlukene kusukela phezulu kuya phansi noma phansi kuya phezulu. Ukwakheka kanye nobukhulu be-gradient medium kukhethwa ngokucophelela, okuqinisekisa ukuthi ilula kune-peroxisomes kodwa iqine kune-lysosomes.

Isinyathelo 2: Ukwendlalela Isampula

Ingxenye ye-lysosome kanye ne-peroxisome ephinde yaxutshwa etholakala ku-differential centrifugation ibekwe ngokucophelela phezu kwe-density gradient medium.

Isinyathelo 3: Ukushintshashintsha kwe-Centrifugation

Ipayipi libe selifakwa ku-centrifugation esheshayo (ultracentrifugation) ukuze kuhlukaniswe ama-lysosome nama-peroxisome ngokusekelwe ekubutheni kwawo. Ngesikhathi sobu-centrifugation, ama-organelle ahamba phakathi kwe-medium aze afike endaweni lapho ukubuthile kwawo kufana khona nokubuthile kwendawo ezungezile, akha amabhande ahlukile.

Ukuhlaziywa kanye Nezicelo Ezengeziwe

Ngemva kokuhlukaniswa okuphumelelayo, izingxenyana ze-lysosome kanye ne-peroxisome ezitholiwe zingahlaziywa kabanzi kusetshenziswa amasu ahlukahlukene ukuhlola ubumsulwa bazo, usayizi, ukwakheka, kanye nezakhiwo zokusebenza. Amasu anjenge-electron microscopy, i-immunocytochemistry, kanye nokuhlolwa komsebenzi we-enzyme anganikeza ukuqonda okubalulekile ngezingxenyana ezihlukanisiwe, okuqinisekisa ubunjalo bazo kanye nobumsulwa bazo.

Ngaphandle kwezinhloso zocwaningo, ikhono lokuhlukanisa ama-lysosome nama-peroxisome ngokusebenzisa i-centrifugation linezinhlelo zokusebenza ekuxilongweni kwezokwelapha, ekuthuthukisweni kwemithi, kanye nasekungeneleleni kokwelapha. Ngokuqonda kangcono imisebenzi yalezi zitho zomzimba, ososayensi bangahlola amasu amasha okwelapha ahlose ukuphazamiseka kwe-lysosomal noma kwe-peroxisomal, njengezifo zokugcina i-lysosomal noma ukuphazamiseka kwe-peroxisome biogenesis.

Ekuphetheni, ukuhlukaniswa kwama-lysosome kuma-peroxisome kusetshenziswa amasu okuhlanganisa, njengokuhlukanisa i-differential centrifugation kanye ne-density gradient centrifugation, kuyindlela ebalulekile ocwaningweni lwebhayoloji yamaseli. Lawa masu avumela ososayensi ukuthola izingxenyana ezihlanziwe zama-lysosome nama-peroxisome, okusiza ekuqondeni izindima zabo ngabanye, izindlela ze-metabolic, kanye nokungenelela okungenzeka kokwelapha izifo ezihlobene.