Kushandiswa uye kungwarira kwe ultrafiltration centrifuge chubhu
1) Sarudza yakakodzera ultrafiltration chubhu.Ziva kuti maUF membranes anosiyana muyero yavo yekushivirira kune akasiyana makemikari.Kazhinji, machubhu e-ultrafiltration ane 10 kDa molecular weight cut-off anogona kusarudzwa ane molecular weight cut-off iyo isingafaniri kuva yakakura kudarika 1/3 yehuremu hwemapuroteni ekufarira, se35 kDa.Kana huremu hwemamolecular eprotein yekufarira iri kutenderera gumi kd, ultrafiltration chubhu ine molecular huremu yakachekwa ye3 KD inogona kushandiswa.
(2) Iyo ultrafiltration, ichangobva kutengwa, yakaoma, ine ultrapure mvura yakawedzerwa isati yashandiswa uye mvura yakanyatsopfuura nepakati pe membrane, ice bath kana firiji pre yakatonhodzwa kwemaminitsi mashoma.Mvura inobva yadirwa, kureva mvura yeprotein, uye inowedzerwa yakawanda sei, chero isingapfuuri mutsetse muchena uri pamusoro pechubhu.Kuvhiya kwakareruka, uye iyo ultrafiltration chubhu inoda kuvharwa pachando isati yawedzera mhinduro yeprotein.
3) Zvose huremu nepakati pegiravhiti zvaifanira kusvika pachiyero.Cherechedza kukurumidza kutenderera uye kukurumidza hakuna kukurumidza, kana zvisina kudaro kukuvadza zvakananga iyo ultrafiltration membrane.Centrifugal ultrafiltration yakatangwa (centrifuge precooled kusvika 4 degrees).Mushure mekunge RPM yemacentrifuges akasiyana akashandurwa kuita g, zvaive zvakasiyana.Kukurumidza kwe centrifuge kwakagadziriswa kusvika padiki, kuderedza kudzvinyirira pa membrane.Ziva, iva nechokwadi chekusiya centrifuge mushure mekunge centrifuge yasvika kumhanya kwairi kuenda, kana kana uine dambudziko, haugone kuribata kekutanga.Kutarisa kwe membrane kune spindle kwakagadziriswa maererano nemirairo (angular centrifuge kesi ndeye membrane kune axis perpendicular).Mukushandiswa kunoshanda, kukurumidza kutenderera kwese kwakaderera pane iyo mumirayiridzo, kuitira kuti hupenyu hwe centrifuge tube huwedzere.
3) Zvose huremu nepakati pegiravhiti zvaifanira kusvika pachiyero.Cherechedza kukurumidza kutenderera uye kukurumidza hakuna kukurumidza, kana zvisina kudaro kukuvadza zvakananga iyo ultrafiltration membrane.Centrifugal ultrafiltration yakatangwa (centrifuge precooled kusvika 4 degrees).Mushure mekunge RPM yemacentrifuges akasiyana akashandurwa kuita g, zvaive zvakasiyana.Kukurumidza kwe centrifuge kwakagadziriswa kusvika padiki, kuderedza kudzvinyirira pa membrane.Ziva, iva nechokwadi chekusiya centrifuge mushure mekunge centrifuge yasvika kumhanya kwairi kuenda, kana kana uine dambudziko, haugone kuribata kekutanga.Kutarisa kwe membrane kune spindle kwakagadziriswa maererano nemirairo (angular centrifuge kesi ndeye membrane kune axis perpendicular).Mukushandiswa kunoshanda, kukurumidza kutenderera kwese kwakaderera pane iyo mumirayiridzo, kuitira kuti hupenyu hwe centrifuge tube huwedzere.
(4) Kana yakanyanyisa kune yakasara 1ml, tora 50ul yebuffer solution, wedzera 10ul yekuyerera uye ona kana pane chero ruvara rwebhuruu, sekutonga kana iyo ultrafiltration chubhu inoshaya mapuroteni.Kana chubhu yadeuka, dururazve denga repamusoro uye woyerera uchienda muchubhu itsva kuti utange kufefetera.Kuti uone chaizvo kana machubhu akapotswa, centrifuge ye10min ine 5mgml yeBSA isati yayerera, ichimhanya paprotein glue, kana Bradford crude assay, uye enderera mberi nekuwedzera yakasara concentrated protein solution (iyo inoshanda pachando uye inodzivirira mapuroteni kubva pakupisa) kusvikira. iyo concentrate yese yawedzerwa.Chenjerera panguva yecentrifugation kana kunaya kwemapuroteni kuchiitika, zvichitungamira kuvharwa kwechubhu.Kana kunaya kukaitika, tarisa kana chikonzero chaicho chekunaya kuri kuwandisa kweprotein kana kuti buffer isina kufanira;Iyo yekutanga inogona kugadziriswa ne ultrafiltration yeakawanda ultrafiltration machubhu panguva imwe chete, kuderedza kusungirirwa, uye yekupedzisira nekuchinjana akasiyana mabuffer mhinduro kudzamara pasina kunaya kweprotein kunoitika.
(4) Kana yakanyanyisa kune yakasara 1ml, tora 50ul yebuffer solution, wedzera 10ul yekuyerera uye ona kana pane chero ruvara rwebhuruu, sekutonga kana iyo ultrafiltration chubhu inoshaya mapuroteni.Kana chubhu yadeuka, dururazve denga repamusoro uye woyerera uchienda muchubhu itsva kuti utange kufefetera.Kuti uone chaizvo kana machubhu akapotswa, centrifuge ye10min ine 5mgml yeBSA isati yayerera, ichimhanya paprotein glue, kana Bradford crude assay, uye enderera mberi nekuwedzera yakasara concentrated protein solution (iyo inoshanda pachando uye inodzivirira mapuroteni kubva pakupisa) kusvikira. iyo concentrate yese yawedzerwa.Chenjerera panguva yecentrifugation kana kunaya kwemapuroteni kuchiitika, zvichitungamira kuvharwa kwechubhu.Kana kunaya kukaitika, tarisa kana chikonzero chaicho chekunaya kuri kuwandisa kweprotein kana kuti buffer isina kufanira;Iyo yekutanga inogona kugadziriswa ne ultrafiltration yeakawanda ultrafiltration machubhu panguva imwe chete, kuderedza kusungirirwa, uye yekupedzisira nekuchinjana akasiyana mabuffer mhinduro kudzamara pasina kunaya kweprotein kunoitika.
(5) Matanho mashoma ekutanga anoshandiswa kuisa puroteni, uye kana buffer ichizoshandurwa, zvinyoronyoro wedzera buffer nyowani (ultrafiltration kuburikidza ne 0.22um ultrafiltration membrane) kusvika ku1ml yeprotein yakazara, uye simbisazve kusvika ku1ml kwevatatu. nguva dzakatevedzana, neiyo yekupedzisira yakanamirwa vhoriyamu yekupedzisira zvichienderana neinodiwa mapuroteni, kazhinji isingapfuuri 500ul, asiwo mukati me200ul.Wana 1000 × kana kupfuura pazviitiko zvitatu, zvakanyanya seyakawanda shanduko yebhafa, sekuverengerwa neinosvika gumi × kana zvakadaro kuwedzera vhoriyamu nguva yega yega.
(5) Matanho mashoma ekutanga anoshandiswa kuisa puroteni, uye kana buffer ichizoshandurwa, zvinyoronyoro wedzera buffer nyowani (ultrafiltration kuburikidza ne 0.22um ultrafiltration membrane) kusvika ku1ml yeprotein yakazara, uye simbisazve kusvika ku1ml kwevatatu. nguva dzakatevedzana, neiyo yekupedzisira yakanamirwa vhoriyamu yekupedzisira zvichienderana neinodiwa mapuroteni, kazhinji isingapfuuri 500ul, asiwo mukati me200ul.Wana 1000 × kana kupfuura pazviitiko zvitatu, zvakanyanya seyakawanda shanduko yebhafa, sekuverengerwa neinosvika gumi × kana zvakadaro kuwedzera vhoriyamu nguva yega yega.
Nguva yekutumira: Nov-09-2022