Ukusetyenziswa kunye nezilumkiso zetyhubhu ye-ultrafiltration centrifuge
1) Khetha ityhubhu ye-ultrafiltration efanelekileyo.Qaphela ukuba ii-membrane ze-UF ziyahluka kwinqanaba labo lokunyamezela kwiikhemikhali ezahlukeneyo.Ngokuqhelekileyo, iityhubhu ze-ultrafiltration ezine-10 kDa ze-molecular weight cut-off zingakhethwa kunye ne-molecular weight cut-off engafanele ibe nkulu kune-1 / 3 ye-molecular weight ye-protein enomdla, njenge-35 kDa.Ukuba ubunzima bemolekyuli yeprotheyini yomdla ijikeleze i-10 kd, ityhubhu ye-ultrafiltration ene-molecular weight cut-off ye-3 KD ingasetyenziswa.
2Amanzi ke agalelwe, okt ulwelo lweproteni, kwaye ingakanani into eyongeziweyo, nokuba yeyiphi engekho ngaphezu komgca omhlophe ongaphezulu kombhobho.Umsebenzi ulula, kwaye ityhubhu ye-ultrafiltration idinga ukucoliswa kwangaphambili kumkhenkce ngaphambi kokongeza isisombululo seprotheni.
3) Zombini ubunzima kunye nombindi womxhuzulane kwakufuneka ufikelele kwibhalansi.Qaphela isantya sokujikeleza kunye nokukhawuleza akukhawulezi kakhulu, ngaphandle koko kuwonakalisa ngokuthe ngqo i-membrane ye-ultrafiltration.I-centrifugal ultrafiltration yaqalwa (i-centrifuge ifakwe ngaphambili kwi-4 degrees).Emva kokuba i-RPM yee-centrifuges ezahlukeneyo iguqulelwe kwi-g, yayihluke.Ukukhawuleza kwe-centrifuge kwahlengahlengiswa ubuncinci, ukunciphisa uxinzelelo kwi-membrane.Qaphela, qiniseka ukuba uyayishiya i-centrifuge emva kokuba i-centrifuge ifikelele kwisantya sayo, okanye ukuba unengxaki, awukwazi ukuyiphatha okokuqala.Ukuqhelaniswa kwe-membrane kwi-spindle kwahlengahlengiswa ngokwemiyalelo (i-angular centrifuge case yi-membrane ukuya kwi-axis perpendicular).Ekusetyenzisweni okusebenzayo, isantya sokujikeleza ngokubanzi sisezantsi kunoko kwimiyalelo, ukwenzela ukuba ubomi bombhobho we-centrifuge bunokwandiswa.
3) Zombini ubunzima kunye nombindi womxhuzulane kwakufuneka ufikelele kwibhalansi.Qaphela isantya sokujikeleza kunye nokukhawuleza akukhawulezi kakhulu, ngaphandle koko kuwonakalisa ngokuthe ngqo i-membrane ye-ultrafiltration.I-centrifugal ultrafiltration yaqalwa (i-centrifuge ifakwe ngaphambili kwi-4 degrees).Emva kokuba i-RPM yee-centrifuges ezahlukeneyo iguqulelwe kwi-g, yayihluke.Ukukhawuleza kwe-centrifuge kwahlengahlengiswa ubuncinci, ukunciphisa uxinzelelo kwi-membrane.Qaphela, qiniseka ukuba uyayishiya i-centrifuge emva kokuba i-centrifuge ifikelele kwisantya sayo, okanye ukuba unengxaki, awukwazi ukuyiphatha okokuqala.I-orientation ye-membrane kwi-spindle yahlengahlengiswa ngokwemiyalelo (i-angular centrifuge case yi-membrane ukuya kwi-axis perpendicular).Ekusetyenzisweni okusebenzayo, isantya sokujikeleza ngokubanzi sisezantsi kunoko kwimiyalelo, ukwenzela ukuba ubomi bombhobho we-centrifuge bunokwandiswa.
4Ukuba ityhubhu iyavuza, phinda ugalele umaleko ongaphezulu kwaye uqukuqelele kwityhubhu entsha ukuqalisa ukuhluza.Ukumisela ngokuchanekileyo ukuba iityhubhu ziphosakele, i-centrifuge ye-10min kunye ne-5mgml ye-BSA ngaphambi kokuhamba, iqhutywe kwiprotheyini yeglue, okanye i-Bradford crude assay, kwaye uqhubeke ngokudibanisa isisombululo seprotheyini eseleyo (esebenza kumkhenkce kwaye inqande iiprotheni ekufudumeni) de yonke into yokugxila yongeziwe.Qaphela ngexesha le-centrifugation ukuba imvula yeeprotheni iyenzeka, ekhokelela ekuvaleni ityhubhu.Ukuba imvula iyenzeka, misela ukuba unobangela othile wemvula kukuxinana kweprotheyini egqithisileyo okanye isithinteli esingafanelekanga;Eyangaphambili ingasonjululwa nge-ultrafiltration yeetyhubhu ezininzi ze-ultrafiltration ngaxeshanye, ukunciphisa ukugxininiswa, kunye nokugqibela ngokutshintshisa izisombululo ze-buffer ezahlukeneyo de kungabikho mvula yeprotheni eyenzekayo.
4Ukuba ityhubhu iyavuza, phinda ugalele umaleko ongaphezulu kwaye uqukuqelele kwityhubhu entsha ukuqalisa ukuhluza.Ukumisela ngokuchanekileyo ukuba iityhubhu ziphosakele, i-centrifuge ye-10min kunye ne-5mgml ye-BSA ngaphambi kokuhamba, iqhutywe kwiprotheyini yeglue, okanye i-Bradford crude assay, kwaye uqhubeke ngokudibanisa isisombululo seprotheyini eseleyo (esebenza kumkhenkce kwaye inqande iiprotheni ekufudumeni) de yonke into yokugxila yongeziwe.Qaphela ngexesha le-centrifugation ukuba imvula yeeprotheni iyenzeka, ekhokelela ekuvaleni ityhubhu.Ukuba imvula iyenzeka, misela ukuba unobangela othile wemvula kukuxinana kweprotheyini egqithisileyo okanye isithinteli esingafanelekanga;Eyangaphambili ingasonjululwa nge-ultrafiltration yeetyhubhu ezininzi ze-ultrafiltration ngaxeshanye, ukunciphisa i-concentration, kunye neyokugqibela ngokutshintshisa izisombululo ze-buffer ezahlukeneyo de kungabikho mvula yeprotheni eyenzekayo.
(5) Amanyathelo okuqala ambalwa asetyenziselwa ukugxila kwiprotheni, kwaye ukuba i-buffer iza kutshintshwa, ngononophelo yongeza i-buffer entsha (i-ultrafiltration nge-0.22um ultrafiltration membrane) malunga ne-1ml yeprotheni iyonke, kwaye uphinde ugxininise malunga ne-1ml ezintathu. amaxesha alandelelanayo, kunye nomthamo wokugqibela ogxininisiweyo wokugqibela ngokuxhomekeke kugxininiso lweprotheyini efunwayo, ngokuqhelekileyo kungekhona ngaphezu kwe-500ul, kodwa nangaphakathi kwe-200ul.Ukuzuza i-1000 × okanye ngaphezulu ngezihlandlo ezithathu, ngokusisiseko njengotshintsho lwe-buffer oluninzi, njengoko kubalwa ubuncinane nge-10 × okanye ngokomthamo wokutyebisa ixesha ngalinye.
(5) Amanyathelo okuqala ambalwa asetyenziselwa ukugxila kwiprotheni, kwaye ukuba i-buffer iza kutshintshwa, ngononophelo yongeza i-buffer entsha (i-ultrafiltration nge-0.22um ultrafiltration membrane) malunga ne-1ml yeprotheni iyonke, kwaye uphinde ugxininise malunga ne-1ml ezintathu. amaxesha alandelelanayo, kunye nomthamo wokugqibela ogxininisiweyo wokugqibela ngokuxhomekeke kugxininiso lweprotheyini efunwayo, ngokuqhelekileyo kungekhona ngaphezu kwe-500ul, kodwa nangaphakathi kwe-200ul.Ukuzuza i-1000 × okanye ngaphezulu ngezihlandlo ezithathu, ngokusisiseko njengotshintsho lwe-buffer oluninzi, njengoko kubalwa ubuncinane nge-10 × okanye ngokomthamo wokutyebisa ixesha ngalinye.
Ixesha lokuposa: Nov-09-2022