Isimiso Sokulinga
I-Hemoglobin electrophoresis ihlose ukuthola nokuqinisekisa ama-hemoglobin ahlukahlukene avamile nangavamile.
Ngenxa yamashaji ahlukene kanye namaphuzu e-isoelectric ezinhlobo ezahlukene ze-hemoglobin, kusixazululo esithile se-pH sebhafa, lapho iphuzu le-isoelectric le-hemoglobin liphansi kune-pH yesisombululo se-buffer, i-hemoglobin ithwala inkokhiso engemihle futhi ifudukela ku-anode ngesikhathi se-electrophoresis. Ngakolunye uhlangothi, i-hemoglobin ene-charge enhle iya ku-cathode.
Ngaphansi kwamandla kagesi athile nangemva kwesikhathi esithile se-electrophoresis, ama-hemoglobin anamacala ahlukene nezisindo zamangqamuzana abonisa izikhombisi-ndlela ezihlukene zokufuduka nesivinini. Lokhu kuvumela ukuhlukaniswa kwezindawo ezihlukene, futhi ukuhlaziywa okulandelayo kwe-colorimetric noma i-electrophoretic scanning kungenziwa kulezi zindawo ukuze kulinganiswe inani lama-hemoglobin ahlukahlukene. Indlela esetshenziswa kakhulu yi-pH 8.6 cellulose acetate membrane electrophoresis.
Ngaphakathi kwe-cytoplasm, amaqembu e-ethylene glycol (CHOH-CHOH) akhona ku-glycogen noma ezintweni ze-polysaccharide (ezifana ne-mucopolysaccharides, mucoproteins, glycoproteins, glycolipids, njll.) i-oxidized by periodic acid futhi iguqulwa ibe amaqembu e-aldehyde (CHO-CHO). Lawa maqembu e-aldehyde ahlangana ne-reagent ye-Schiff engenambala onsomi-bomvu, akhe udayi onsomi-obomvu ofaka lapho ama-polysaccharides ekhona kuseli. Lokhu kusabela kwaziwa ngokuthi i-periodic acid-Schiff (PAS) staining, ngaphambilini ebibizwa ngokuthi i-glycogen staining.
Indlela Yokuhlola
Izinto zokwakha:I-Cellulose acetatememi-brane, i-electrophoresis apparatus(I-DYCP-38C namandla kagesi i-DYY-6C), Ithuluzi Lokulayisha Isampula Eliphezulu(i-pipette), i-spectrophotometer, i-colorimetric cuvettes, ama-buffers.
Isigcinalwazi:
(1) pH 8.6 TEB Buffer: Kala 10.29 g Tris, 0.6 g EDTA, 3.2 g boric acid, bese wengeza amanzi distilled ku-1000 ml.
(2) I-Borate Buffer: Kala u-6.87 g we-borax no-5.56 g we-boric acid, bese wengeza amanzi acwecwe ku-1000 ml.
Inqubo:
Pukulungiswa kweHemoglobin Solution
Thatha u-3 ml wegazi eliqukethe i-heparin noma i-sodium citrate njenge-anticoagulant. I-Centrifuge ku-2000 rpm imizuzu engu-10 bese ulahla i-plasma. Geza amangqamuzana abomvu egazi kathathu nge-saline ye-physiological (750 rpm, imizuzu emi-5 ye-centrifugation isikhathi ngasinye). I-Centrifuge ku-2200 rpm imizuzu engu-10 bese ulahla i-supernatant. Engeza inani elilinganayo lamanzi acwecwe, bese wengeza izikhathi ezingu-0.5 zevolumu ye-carbon tetrachloride. Shake ngamandla imizuzu emi-5, bese u-centrifuge ku-2200 rpm imizuzu eyi-10 ukuqoqa isisombululo se-Hb esiphezulu ukuze sisetshenziswe kamuva.
Ukucwilisa iMembrane
Sika ulwelwesi lwe-cellulose acetate lube yimicu engu-3 cm × 8 cm. Wacwilise ku-pH 8.6 TEB buffer aze agcwale ngokugcwele, bese uyawasusa bese uwasula ngephepha lokuhlunga.
Ukubona
Sebenzisa i-pipette ukuze ubone u-10 μl wesisombululo se-hemoglobin ubheke phezulu kulwelwesi lwe-cellulose acetate (uhlangothi olumaholoholo), cishe u-1.5 cm ukusuka onqenqemeni.
I-Electrophoresis
Thela isisombululo se-borate buffer ekamelweni le-electrophoresis. Beka ulwelwesi lwe-cellulose acetate ohlangothini olunamachashaza ekugcineni kwe-cathode yegumbi. Bhaka ku-200 V imizuzu engu-30.
I-Elution
Sika izindawo ze-HbA ne-HbA2, uzibeke kumashubhu okuhlola ahlukene, bese wengeza u-15 ml no-3 ml wamanzi acwecwe, ngokulandelana. Nyakazisa kancane ukuze ukhiphe i-hemoglobin ngokuphelele, bese uxuba.
I-Colorimetry
Zero ukumunca usebenzisa amanzi acwecwe ukuze kuxazululwe isixazululo bese ukala ukumunca ku-415 nm.
Ukubala
I-HbA2(%) = Ukungabi bikho kweshubhu le-HbA2 / (Ukungabi khona kweshubhu le-HbA × 5 + Ukungabikho kweshubhu le-HbA2) × 100%
Ukubalwa Kwemiphumela Yokuhlola
Ireferensi Ububanzi be-pH 8.6 TEB Buffer Cellulose Acetate Electrophoresis: HbA > 95%, HbA2 1% -3.1%
Amanothi
Isikhathi se-Electrophoresis akufanele sibe side kakhulu. I-cellulose acetate membrane akufanele yome ngesikhathi se-electrophoresis. Misa i-electrophoresis lapho i-HbA ne-HbA2 zihlukaniswa ngokucacile. I-electrophoresis ende ingabangela ukusakazeka kwebhande nokufiphala.
Gwema ukusebenzisa amasampula amaningi kakhulu. Uketshezi lwe-hemoglobin eyeqile lungaholela ekuhlukanisweni kwebhande noma ukungcola okwanele, okuholela ekukhuphukeni kwamazinga e-HbA ngokungeyikho.
Vimbela ukungcoliswa kwe-cellulose acetate membrane ngamaprotheni.
Umsinga akufanele ube phezulu kakhulu; ngaphandle kwalokho, amabhande e-hemoglobin angeke ahlukane.
Hlala uhlanganisa izifanekiso ezivela kubantu abajwayelekile kanye nama-hemoglobin adingekayo awaziwayo njengezilawuli.
I-Beijing Liuyi Biotechnology yenza ithangi le-electrophoresis lochwepheshe le-hemoglobin electrophoresis okuyimodeliI-DYCP-38Cithangi le-cellulose acetate membrane electrophoresis tank, futhi kukhona amamodeli amabili okunikezwa kwamandla e-electrophoresis atholakalayo kuthangi le-cellulose acetate membrane electrophoresis tank.I-DYY-2CfuthiI-DYY-6Cukunikezwa kwamandla kagesi.
Ngaleso sikhathi, i-Beijing Liuyi Biotechnology inikeza ulwelwesi lwe-cellulose acetate kumakhasimende, futhi usayizi we-cellulose acetate membrane ungenziwa ngokwezifiso. Siyakwamukela ukusibuza amasampula kanye nolwazi olwengeziwe.
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Isikhathi sokuthumela: Sep-20-2023