Kugwiritsa ntchito ndi kusamala kwa ultrafiltration centrifuge chubu
1) Sankhani chubu choyenera cha ultrafiltration.Zindikirani kuti nembanemba za UF zimasiyana pakulekerera kwawo mankhwala osiyanasiyana.Nthawi zambiri, machubu a ultrafiltration okhala ndi 10 kDa molecular weight cut-off amatha kusankhidwa ndi kudulidwa kwa mamolekyulu omwe sayenera kukhala wamkulu kuposa 1/3 ya kulemera kwa ma protein omwe amafunikira, monga 35 kDa.Ngati kulemera kwa molekyulu ya protein yosangalatsa ndi pafupifupi 10 kd, chubu cha ultrafiltration chokhala ndi mamolekyulu odulidwa a 3 KD angagwiritsidwe ntchito.
(2) The ultrafiltration, mwatsopano anagula, ndi youma, ndi ultrapure madzi anawonjezera pamaso ntchito ndi madzi kwathunthu kudutsa nembanemba, ayezi kusamba kapena firiji pre utakhazikika kwa mphindi zingapo.Madziwo amatsanulidwa, mwachitsanzo, madzi a puloteni, ndi kuchuluka kwake komwe amawonjezeredwa, kaya ndi chiyani chomwe sichiposa mzere woyera pamwamba pa chubu.Opaleshoniyo ndi yopepuka, ndipo chubu cha ultrafiltration chiyenera kuziziritsidwa pa ayezi musanawonjezere yankho la mapuloteni.
3) Unyinji ndi pakati pa mphamvu yokoka zimayenera kufika pamlingo.Dziwani kuti liwiro lozungulira komanso kuthamanga sikuthamanga kwambiri, mwinanso kuwononga nembanemba ya ultrafiltration.Centrifugal ultrafiltration idayambika (centrifuge precooled to 4 degrees).Pambuyo pa RPM ya ma centrifuges osiyanasiyana adasinthidwa kukhala g, zinali zosiyana.Kuthamanga kwa centrifuge kunasinthidwa kukhala kochepa, kuchepetsa kupanikizika pa nembanemba.Zindikirani, onetsetsani kuti mwasiya centrifuge centrifuge ikafika pa liwiro lomwe likupita, kapena ngati muli ndi vuto, simungathe kuigwira kwa nthawi yoyamba.Mayendedwe a nembanemba ku spindle adasinthidwa molingana ndi malangizo (angular centrifuge kesi ndi membrane to axis perpendicular).Pogwiritsira ntchito, kuthamanga kwachangu kumakhala kochepa kusiyana ndi malangizo, kuti moyo wa chubu la centrifuge uwonjezeke.
3) Unyinji ndi pakati pa mphamvu yokoka zimayenera kufika pamlingo.Dziwani kuti liwiro lozungulira komanso kuthamanga sikuthamanga kwambiri, mwinanso kuwononga nembanemba ya ultrafiltration.Centrifugal ultrafiltration idayambika (centrifuge precooled to 4 degrees).Pambuyo pa RPM ya ma centrifuges osiyanasiyana adasinthidwa kukhala g, zinali zosiyana.Kuthamanga kwa centrifuge kunasinthidwa kukhala kochepa, kuchepetsa kupanikizika pa nembanemba.Zindikirani, onetsetsani kuti mwasiya centrifuge centrifuge ikafika pa liwiro lomwe likupita, kapena ngati muli ndi vuto, simungathe kuigwira kwa nthawi yoyamba.Mayendedwe a nembanemba ku spindle adasinthidwa molingana ndi malangizo (angular centrifuge kesi ndi membrane to axis perpendicular).Pogwiritsira ntchito, kuthamanga kwachangu kumakhala kochepa kusiyana ndi malangizo, kuti moyo wa chubu la centrifuge uwonjezeke.
(4) Mukakhazikika ku 1ml yotsalayo, tengani 50ul ya yankho la buffer, onjezerani 10ul yothamanga ndikuwona ngati pali mtundu wa buluu, monga chigamulo ngati chubu cha ultrafiltration chikusowa mapuloteni.Ngati chubu latopa, tsitsaninso gawo lapamwamba ndikulowa mu chubu chatsopano kuti muyambitse kusefera.Kuti mudziwe ndendende ngati machubu anaphonya, centrifuge kwa 10min ndi 5mgml wa BSA isanafike flowthrough, kuthamanga pa mapuloteni guluu, kapena Bradford crude assay, ndi kupitiriza ndi kuwonjezera otsala anaikira mapuloteni njira (yomwe imagwira ntchito pa ayezi ndi kuteteza mapuloteni kutentha) mpaka zonse zokhazikika zawonjezedwa.Samalani panthawi ya centrifugation ngati mpweya wa mapuloteni umachitika, zomwe zimapangitsa kuti chubu litseke.Ngati mvula igwa, dziwani ngati chomwe chikuyambitsa mvula ndi kuchuluka kwa mapuloteni ochulukirapo kapena chitetezo chosayenera;Zoyambazo zitha kuthetsedwa ndi kusefedwa kwa machubu angapo a ultrafiltration nthawi imodzi, kutsitsa ndende, ndipo komalizako posinthana njira zosiyanasiyana zosungira mpaka mpweya wa protein usagwe.
(4) Mukakhazikika ku 1ml yotsalayo, tengani 50ul ya yankho la buffer, onjezerani 10ul yothamanga ndikuwona ngati pali mtundu wa buluu, monga chigamulo ngati chubu cha ultrafiltration chikusowa mapuloteni.Ngati chubu latopa, tsitsaninso gawo lapamwamba ndikulowa mu chubu chatsopano kuti muyambitse kusefera.Kuti mudziwe ndendende ngati machubu anaphonya, centrifuge kwa 10min ndi 5mgml wa BSA isanafike flowthrough, kuthamanga pa mapuloteni guluu, kapena Bradford crude assay, ndi kupitiriza ndi kuwonjezera otsala anaikira mapuloteni njira (yomwe imagwira ntchito pa ayezi ndi kuteteza mapuloteni kutentha) mpaka zonse zokhazikika zawonjezedwa.Samalani panthawi ya centrifugation ngati mpweya wa mapuloteni umachitika, zomwe zimapangitsa kuti chubu litseke.Ngati mvula igwa, dziwani ngati chomwe chikuyambitsa mvula ndi kuchuluka kwa mapuloteni ochulukirapo kapena chitetezo chosayenera;Zoyambazo zitha kuthetsedwa ndi kusefedwa kwa machubu angapo a ultrafiltration nthawi imodzi, kutsitsa kuchulukira, ndipo komalizako posinthana njira zosiyanasiyana zosungira mpaka mpweya wa protein usagwe.
(5) Njira zingapo zoyamba zimagwiritsidwa ntchito kuyika puloteni, ndipo ngati chotchingacho chiyenera kusinthidwa, onjezerani pang'onopang'ono chotchinga chatsopano (ultrafiltration kupyolera mu 0.22um ultrafiltration nembanemba) mpaka pafupifupi 1ml ya mapuloteni okwana, ndi kubwereranso ku 1ml kwa atatu. nthawi zotsatizana, ndi voliyumu yomaliza yokhazikika kutengera kuchuluka kwa mapuloteni omwe amafunidwa, nthawi zambiri osapitilira 500ul, komanso mkati mwa 200ul.Fikirani 1000 × kapena kupitilira apo katatu, makamaka kusintha kocheperako, monga kuwerengeredwa ndi kuchuluka kwa 10 × kapena kupitilira apo nthawi iliyonse.
(5) Njira zingapo zoyamba zimagwiritsidwa ntchito kuyika puloteni, ndipo ngati chotchingacho chiyenera kusinthidwa, onjezerani pang'onopang'ono chotchinga chatsopano (ultrafiltration kupyolera mu 0.22um ultrafiltration nembanemba) mpaka pafupifupi 1ml ya mapuloteni okwana, ndi kubwereranso ku 1ml kwa atatu. nthawi zotsatizana, ndi voliyumu yomaliza yokhazikika kutengera kuchuluka kwa mapuloteni omwe amafunidwa, nthawi zambiri osapitilira 500ul, komanso mkati mwa 200ul.Fikirani 1000 × kapena kupitilira apo katatu, makamaka kusintha kocheperako, monga kuwerengeredwa ndi kuchuluka kwa 10 × kapena kupitilira apo nthawi iliyonse.
Nthawi yotumiza: Nov-09-2022