Ukusetshenziswa kanye nezixwayiso ze-ultrafiltration centrifuge tube
1) Khetha ishubhu ye-ultrafiltration efanelekile.Qaphela ukuthi ama-membrane e-UF ayahluka ngezinga lawo lokubekezelela amakhemikhali ahlukahlukene.Ngokuvamile, amashubhu e-ultrafiltration ane-10 kDa isisindo esinqunyiwe angakhethwa ngokusikwa kwesisindo se-molecular okungafanele sibe sikhulu kuno-1/3 wesisindo se-molecular of the protein of interest, njenge-35 kDa.Uma isisindo semolekyuli sephrotheni esithakaselwayo singaba ngu-10 kd, ishubhu yokuhlunga nge-molecular weight cut-off engu-3 KD ingasetshenziswa.
(2) I-ultrafiltration, esanda kuthengwa, yomile, inamanzi ahlanzekile engezwe ngaphambi kokusetshenziswa futhi amanzi adlule ngokuphelele kulwelwesi, okugeza kweqhwa noma isiqandisi esipholile ngaphambi kwemizuzu embalwa.Amanzi abe esethelwa, okungukuthi uketshezi lweprotheyini, nokuthi kungezwa malini, noma yikuphi okungengaphezu komugqa omhlophe phezulu kweshubhu.Ukusebenza kulula, futhi ishubhu lokuhlunga lidinga ukupholiswa kusengaphambili eqhweni ngaphambi kokwengeza isisombululo seprotheni.
3) Kokubili ubukhulu kanye nendawo yamandla adonsela phansi kwakufanele kufinyelele esilinganisweni.Qaphela isivinini sokujikeleza nokusheshisa akusheshi kakhulu, ngaphandle kwalokho kulimaza ngokuqondile ulwelwesi lwe-ultrafiltration.I-centrifugal ultrafiltration yaqalwa (i-centrifuge ifakwe kuqala ku-4 degrees).Ngemuva kokuthi i-RPM yama-centrifuges ahlukene iguqulelwe ku-g, ibihlukile.Ukusheshisa kwe-centrifuge kwalungiswa okungenani, kunciphisa ukucindezela kulwelwesi.Qaphela, qiniseka ukuthi uyayishiya i-centrifuge ngemva kokuba i-centrifuge isifinyelele isivinini lapho iya khona, noma uma unenkinga, awukwazi ukuyiphatha okokuqala.Ukuma kwe-membrane ku-spindle kwalungiswa ngokulandela imiyalelo (i-angular centrifuge case i-membrane kuya ku-axis perpendicular).Ekusetshenzisweni okungokoqobo, ijubane elijwayelekile lokujikeleza liphansi kunalelo emiyalweni, ukuze impilo yeshubhu ye-centrifuge inwetshwe.
3) Kokubili ubukhulu kanye nendawo yamandla adonsela phansi kwakufanele kufinyelele esilinganisweni.Qaphela isivinini sokujikeleza nokusheshisa akusheshi kakhulu, ngaphandle kwalokho kulimaza ngokuqondile ulwelwesi lwe-ultrafiltration.I-centrifugal ultrafiltration yaqalwa (i-centrifuge ifakwe kuqala ku-4 degrees).Ngemuva kokuthi i-RPM yama-centrifuges ahlukene iguqulelwe ku-g, ibihlukile.Ukusheshisa kwe-centrifuge kwalungiswa okungenani, kunciphisa ukucindezela kulwelwesi.Qaphela, qiniseka ukuthi uyayishiya i-centrifuge ngemva kokuba i-centrifuge isifinyelele isivinini lapho iya khona, noma uma unenkinga, awukwazi ukuyiphatha okokuqala.Ukuma kwe-membrane ku-spindle kwalungiswa ngokulandela imiyalelo (i-angular centrifuge case i-membrane kuya ku-axis perpendicular).Ekusetshenzisweni okungokoqobo, ijubane elijwayelekile lokujikeleza liphansi kunalelo emiyalweni, ukuze impilo yeshubhu ye-centrifuge inwetshwe.
(4) Uma ugxiliswe ku-1ml esele, thatha u-50ul wekhambi le-buffer, engeza i-10ul yokugeleza futhi ubone ukuthi awukho yini umbala oluhlaza okwesibhakabhaka, njengesinqumo sokuthi ishubhu lokuhlunga alinawo amaprotheni.Uma ishubhu livuza, phinda uthele ungqimba olungaphezulu bese ugeleza ungene eshubhuni elisha ukuze uqale ukucwenga okukhulu.Ukunquma kahle ukuthi amashubhu abegejiwe yini, i-centrifuge ye-10min ne-5mgml ye-BSA ngaphambi kokugeleza, isebenza nge-protein glue, noma i-Bradford crude assay, futhi uqhubeke ngokungeza isisombululo esisele samaprotheni (esisebenza eqhweni futhi sivimbele amaprotheni ekushiseni) kuze kube yilapho. konke okugxilile kwengeziwe.Nakekela ngesikhathi se-centrifugation uma kunemvula yamaprotheni, okuholela ekuvaleni kwamashubhu.Uma izulu liba khona, nquma ukuthi ingabe imbangela ethile yemvula iwukugcwala kwamaprotheni eqile noma isikhunta esingafanele;Okwangaphambili kungaxazululwa ngokucwenga okukhulu kwamashubhu amaningi okuhlunga ngasikhathi sinye, kwehliswe ukugxilisa ingqondo, futhi lokhu kwakamuva ngokushintshisana ngezisombululo zebhafa ezihlukene kuze kungabi bikho ukuna kwephrotheni.
(4) Uma ugxiliswe ku-1ml esele, thatha u-50ul wekhambi le-buffer, engeza i-10ul yokugeleza futhi ubone ukuthi awukho yini umbala oluhlaza okwesibhakabhaka, njengesinqumo sokuthi ishubhu lokuhlunga alinawo amaprotheni.Uma ishubhu livuza, phinda uthele ungqimba olungaphezulu bese ugeleza ungene eshubhuni elisha ukuze uqale ukucwenga okukhulu.Ukunquma kahle ukuthi amashubhu abegejiwe yini, i-centrifuge ye-10min ne-5mgml ye-BSA ngaphambi kokugeleza, isebenza nge-protein glue, noma i-Bradford crude assay, futhi uqhubeke ngokungeza isisombululo esisele samaprotheni (esisebenza eqhweni futhi sivimbele amaprotheni ekushiseni) kuze kube yilapho. konke okugxilile kwengeziwe.Nakekela ngesikhathi se-centrifugation uma kunemvula yamaprotheni, okuholela ekuvaleni kwamashubhu.Uma izulu liba khona, nquma ukuthi ingabe imbangela ethile yemvula iwukugcwala kwamaprotheni eqile noma isikhunta esingafanele;Okwangaphambili kungaxazululwa ngokucwenga okukhulu kwamashubhu amaningi okuhlunga ngasikhathi sinye, kwehliswe ukugxilisa ingqondo, futhi lokhu kwakamuva ngokushintshisana ngezisombululo zebhafa ezihlukene kuze kungabi bikho ukuna kwephrotheni.
(5) Izinyathelo ezimbalwa zokuqala zisetshenziselwa ukugxilisa iphrotheni, futhi uma isilondolozi sizoshintshwa, engeza ngobumnene isigcinalwazi esisha (i-ultrafiltration ngokusebenzisa ulwelwesi oluyi-ultrafiltration oluyi-0.22um) lube cishe ku-1ml wengqikithi yephrotheni, futhi ugxilise kabusha ku-1ml kokuthathu. izikhathi ezilandelanayo, nevolumu yokugcina egxilile kuye ngokuthi i-protein efunwayo, ngokuvamile ayidluli ku-500ul, kodwa futhi ibe ngaphakathi kwama-200ul.Zuza u-1000 × noma ngaphezulu izikhathi ezintathu, ngokuyisisekelo njengoshintsho lwebhafa oluningi, njengoba kubalwa okungenani ngo-10 × noma ngaphezulu kokunothisa ivolumu isikhathi ngasinye.
(5) Izinyathelo ezimbalwa zokuqala zisetshenziselwa ukugxilisa iphrotheni, futhi uma isilondolozi sizoshintshwa, engeza ngobumnene isigcinalwazi esisha (i-ultrafiltration ngokusebenzisa ulwelwesi oluyi-ultrafiltration oluyi-0.22um) lube cishe ku-1ml wengqikithi yephrotheni, futhi ugxilise kabusha ku-1ml kokuthathu. izikhathi ezilandelanayo, nevolumu yokugcina egxilile kuye ngokuthi i-protein efunwayo, ngokuvamile ayidluli ku-500ul, kodwa futhi ibe ngaphakathi kwama-200ul.Zuza u-1000 × noma ngaphezulu izikhathi ezintathu, ngokuyisisekelo njengoshintsho lwebhafa oluningi, njengoba kubalwa okungenani ngo-10 × noma ngaphezulu kokunothisa ivolumu isikhathi ngasinye.
Isikhathi sokuthumela: Nov-09-2022