indlela yokwahlula ii-lysosomes kwi-peroxisomes nge-centrifugation
Intshayelelo kwiLysosomes kunye nePeroxisomes
IiLysosomes kunye neeperoxisomes zii-organelles ezimbini ezahlukeneyo zeeseli ezinemisebenzi eyahlukileyo edlala indima ebalulekileyo kwimetabolism yeseli kunye ne-homeostasis. IiLysosomes zii-organelles ezibotshelelwe kwi-membrane ezizaliswe zii-enzymes zokugaya ukutya ezinoxanduva lokuqhekeza izinto ezilahliweyo zeseli, amasuntswana angaphandle, kunye neentsholongwane. IiPeroxisomes, kwelinye icala, zibandakanyeka kwiimpendulo ezahlukeneyo zemetabolism, ngakumbi ezo zinxulumene nokukhupha ubuthi kunye nemetabolism ye-lipid. Ukuze sifunde ezi organelles ngokwahlukeneyo, enye indlela esetyenziswa kakhulu yi-centrifugation. Kweli nqaku, siza kuhlola indlela i-centrifugation engasetyenziswa ngayo ukwahlula ngempumelelo ii-lysosomes kwi-peroxisomes.
Imigaqo kunye neendlela zokusebenzisa i-Centrifugation
I-Centrifugation yindlela enamandla esetyenziselwa ukwahlula izinto ezahlukeneyo zomxube owahlukileyo ngokusekelwe kubukhulu bazo, uxinano, kunye nokuma kwazo. Isebenza kwimigaqo ye-sedimentation, apho amasuntswana anzima ahlala khona ngokukhawuleza phantsi kwempembelelo yamandla e-centrifugal. Ukwahlula ii-lysosomes kwi-peroxisomes, iindlela ezahlukeneyo ze-centrifugation, ezifana ne-differential centrifugation kunye ne-density gradient centrifugation, zingasetyenziswa.
Ukwahlulahlula kweCentrifugation: Ukukhupha iiLysosomes kunye neePeroxisomes
I-differential centrifugation ibandakanya uthotho lwamanyathelo e-centrifugation ngesantya esahlukileyo kunye nobude bokwahlula ii-organelles ngokusekelwe kubuninzi bazo. Nantsi indlela engasetyenziswa ngayo ukukhupha ii-lysosomes kunye nee-peroxisomes:
Inyathelo 1: Ukudibanisa
Inyathelo lokuqala kukuqhekeza iiseli kancinci uze ukhulule umxholo wazo usebenzisa indlela yokwenza i-homogenization. Oku kunokufezekiswa ngokufaka i-sonicating okanye ukusebenzisa i-homogenizer ekhethekileyo. Umxube ophumayo ubizwa ngokuba yi-homogenate.
Inyathelo lesi-2: I-Centrifugation ekhawulezayo
I-homogenate emva koko ifakwa kwi-centrifugation enesantya esiphantsi (malunga ne-1,000-2,000 xg) ixesha elifutshane. Eli nyathelo linceda ukwahlula inkunkuma enkulu yeseli kunye nenxalenye yenyukliya kwezinye ii-organelles ezikhoyo kwi-homogenate.
Inyathelo lesi-3: Ukwandisa i-Centrifugation ngesantya esiphakathi
I-supernatant efunyenwe kwinyathelo le-centrifugation eliphantsi ifakwa kwi-centrifugation yesantya esiphakathi (malunga ne-10,000 xg) ukuze ichithe ii-organelles ezinzima ezifana nee-lysosomes kunye nee-peroxisomes. Eli nyathelo liphumela kwi-pellet equlethe ezi organelles.
Inyathelo lesi-4: Hlamba kwaye Uphinde Usebenzise Umoya Ococekileyo
I-pellet efunyenwe kwinyathelo elingaphambili ihlanjwa ngesisombululo esifanelekileyo esifakwe i-buffer kwaye emva koko iphinda ixhonywe ukuze kufunyanwe i-lysosome ecocekileyo kunye ne-peroxisome fraction.
I-Centrifugation ye-Density Gradient: Ukuhlaziya uLwahlulo
I-centrifugation ye-density gradient yindlela ephucukileyo ye-centrifugation evumela ukucocwa nokucocwa ngakumbi kwe-lysosomes kunye ne-peroxisomes. Kule ndlela, i-density gradient yenziwa kusetyenziswa isisombululo sokwenza i-gradient esifana ne-sucrose okanye i-iodixanol. Nantsi indlela engasetyenziswa ngayo:
Inyathelo 1: Ukulungiswa kweGradient yoBuninzi
I-density gradient medium, efana ne-sucrose, ilungiswa kwityhubhu ye-centrifuge, enobunzima obahlukeneyo ukusuka phezulu ukuya ezantsi okanye ezantsi ukuya phezulu. Ulwakhiwo kunye nobunzima be-gradient medium bukhethwa ngononophelo, ukuqinisekisa ukuba ilula kune-peroxisomes kodwa ixinene kune-lysosomes.
Inyathelo lesi-2: Ukubekwa kweSampuli
I-lysosome ephinde yaxutywa kunye ne-peroxisome fraction efunyenwe kwi-differential centrifugation ibekwe ngononophelo phezu kwe-density gradient medium.
Inyathelo lesi-3: Ukubeka i-Centrifugation
Emva koko ityhubhu ifakwa kwi-centrifugation ekhawulezayo (ultracentrifugation) ukuze yahlule ii-lysosomes kunye nee-peroxisomes ngokusekelwe kubuninzi bazo obuthambileyo. Ngexesha le-centrifugation, ii-organelles zihamba nge-medium zide zifike kwindawo apho ubuninzi bazo obuthambileyo buhambelana nobunzima be-medium ejikelezileyo, zenze imida eyahlukileyo.
Uhlalutyo kunye nezinye izicelo
Emva kokwahlulwa ngempumelelo, ii-lysosome kunye nee-peroxisome fractions ezifunyenweyo zinokuhlalutywa ngakumbi kusetyenziswa iindlela ezahlukeneyo zokuvavanya ubunyulu bazo, ubungakanani, ukwakheka kwazo, kunye neempawu zokusebenza kwazo. Iindlela ezifana ne-electron microscopy, i-immunocytochemistry, kunye novavanyo lomsebenzi we-enzyme zinokubonelela ngengcaciso ebalulekileyo kwii-fractions ezizimeleyo, ziqinisekisa ubunyani bazo kunye nobunyulu bazo.
Ngaphandle kweenjongo zophando, ukukwazi ukwahlula ii-lysosomes kunye nee-peroxisomes ngokusebenzisa i-centrifugation kunezicelo zokuxilongwa kwezonyango, uphuhliso lwamayeza, kunye nokungenelela konyango. Ngokuqonda ngcono imisebenzi yezi organelles, izazinzulu zinokuhlola amaqhinga amatsha okunyanga ajolise kwiingxaki ze-lysosomal okanye ze-peroxisomal, ezifana nezifo zokugcina i-lysosomal okanye iingxaki ze-peroxisome biogenesis.
Ukuqukumbela, ukwahlulwa kwee-lysosomes kwi-peroxisomes kusetyenziswa iindlela ze-centrifugation, ezifana ne-differential centrifugation kunye ne-density gradient centrifugation, yindlela ebalulekileyo kuphando lwebhayoloji yeseli. Ezi ndlela zivumela izazinzulu ukuba zifumane amaqhezu acocekileyo e-lysosomes kunye ne-peroxisomes, zinceda ekuqondeni iindima zazo, iindlela ze-metabolic, kunye nokungenelela okunokwenzeka kwezonyango kwizifo ezinxulumene noko.
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