Uvavanyo lweHemoglobin Electrophoresis

UmGaqo wokuLinga

IHemoglobin electrophoresis ijolise ekufumaniseni nasekuqinisekiseni iihemoglobins ezahlukeneyo eziqhelekileyo nezingaqhelekanga.

Ngenxa yeentlawulo ezahlukeneyo kunye namanqaku e-isoelectric eentlobo ezahlukeneyo ze-hemoglobin, kwisisombululo esithile se-pH se-buffer, xa indawo ye-isoelectric ye-hemoglobin ingaphantsi kune-pH yesisombululo se-buffer, i-hemoglobin ithwala intlawulo engalunganga kwaye ifudukela kwi-anode ngexesha le-electrophoresis. Ngakolunye uhlangothi, i-hemoglobin enexabiso elihle ihambela kwi-cathode.

Ngaphantsi kombane othile kunye nasemva kwexesha elithile le-electrophoresis, ii-hemoglobins ezineentlawulo ezahlukeneyo kunye nobunzima beemolekyuli zibonisa iindlela ezahlukeneyo zokufuduka kunye nezantya. Oku kuvumela ukwahlulwa kwemimandla eyahlukeneyo, kwaye uhlalutyo olulandelayo lwe-colorimetric okanye i-electrophoretic scanning lunokwenziwa kule mimandla ukulinganisa ii-hemoglobins ezahlukeneyo. Eyona ndlela isetyenziswa ngokuqhelekileyo yi-pH 8.6 cellulose acetate membrane electrophoresis.

Ngaphakathi kwe-cytoplasm, amaqela e-ethylene glycol (CHOH-CHOH) akhoyo kwi-glycogen okanye kwi-polysaccharide izinto (ezifana ne-mucopolysaccharides, i-mucoproteins, i-glycoproteins, i-glycolipids, njl.) i-oxidized by periodic acid kwaye iguqulelwe kumaqela e-aldehyde (CHO-CHO). La maqela e-aldehyde adibanisa kunye ne-reagent ye-Schiff engenambala e-purplish-red-red, yenza idayi emfusa-bomvu ebeka apho iipolysaccharides zikhona kwiseli. Le ntshukumo yaziwa ngokuba yi-periodic acid-Schiff (PAS) staining, eyayifudula ibizwa ngokuba yi-glycogen staining.

Uvavanyo lweNdlela

Izixhobo:I-cellulose acetatememibrane, izixhobo ze-electrophoresis(DYCP-38C kunye nombane weDYY-6C), Isixhobo sokuLayisha iSampuli esiPhezulu (ipayipi), spectrophotometer, colorimetric cuvettes, izithinteli.

Isithinteli:

(1) pH 8.6 TEB Buffer: Ubunzima 10.29 g Tris, 0.6 g EDTA, 3.2 g boric acid, kwaye wongeze amanzi distilled ukuya 1000 ml.

(2) I-Borate Buffer: Ubunzima be-6.87 g yeboraksi kunye ne-5.56 g ye-boric acid, kwaye wongeze amanzi adibeneyo kwi-1000 ml.

Inkqubo:

Pukulungiswa kweHemoglobin Solution

Thatha i-3 ml yegazi equkethe i-heparin okanye i-sodium citrate njenge-anticoagulant. I-Centrifuge kwi-2000 rpm imizuzu eyi-10 kwaye ulahle i-plasma. Hlamba iiseli ezibomvu zegazi kathathu nge-saline ye-physiological (750 rpm, imizuzu emi-5 ye-centrifugation ngexesha ngalinye). I-Centrifuge kwi-2200 rpm imizuzu eyi-10 kwaye ulahle i-supernatant. Yongeza inani elilinganayo lamanzi adibeneyo, uze udibanise amaxesha angama-0.5 umthamo we-carbon tetrachloride. Shake ngamandla imizuzu emi-5, kwaye emva koko i-centrifuge kwi-2200 rpm imizuzu eyi-10 ukuqokelela isisombululo se-Hb esiphezulu sokusetyenziswa kamva.

Ukumanzisa iMembrane

Sika i-cellulose acetate membrane ibe yimichilo yokulinganisa i-3 cm × 8 cm. Zifunxe kwi-pH 8.6 TEB buffer de zihluthe ngokupheleleyo, uze uzisuse kwaye wome ngephepha lokucoca.

Ukubona

Sebenzisa i-pipette ukubona i-10 μl yesisombululo se-hemoglobin ngokuthe nkqo kwi-cellulose acetate inwebu (icala elirhabaxa), malunga ne-1.5 cm ukusuka emphethweni.

Electrophoresis

Galela isisombululo se-borate buffer kwigumbi le-electrophoresis. Beka i-cellulose acetate membrane kunye necala elibonakalayo ekupheleni kwe-cathode yegumbi. Baleka kwi-200 V imizuzu engama-30.

Uvavanyo

Sika iindawo ze-HbA kunye ne-HbA2, uzibeke kwiibhubhu zokuvavanya ezihlukeneyo, kwaye ungeze i-15 ml kunye ne-3 ml yamanzi adibeneyo, ngokulandelanayo. Gxuma ngobunono ukuze ukhuphe i-hemoglobin ngokupheleleyo, uze udibanise.

Umbala wemibala

Zero i-absorption usebenzisa amanzi adibeneyo kwisisombululo se-elution kwaye ulinganise ukufunxa kwi-415 nm.

Ukubala

I-HbA2 (%) = Ukungabikho kwe-tube ye-HbA2 / (Ukungabikho kwe-tube ye-HbA × 5 + Ukungaphumi kwe-tube ye-HbA2) × 100%

Ubalo lweZiphumo zoMfuniselo

Uluhlu lweeReferensi ze-pH 8.6 TEB Buffer Cellulose Acetate Electrophoresis: HbA > 95%, HbA2 1% -3.1%

Amanqaku

Ixesha le-Electrophoresis akufanele libe lide kakhulu. I-cellulose acetate membrane akufanele yome ngexesha le-electrophoresis. Yeka i-electrophoresis xa i-HbA kunye ne-HbA2 zihlulwe ngokucacileyo. I-electrophoresis ende inokubangela ukusasazwa kwebhendi kunye nokufiphala.

Kuphephe ukusebenzisa isampulu eninzi. Ulwelo olugqithisileyo lwe-hemoglobin lunokukhokelela ekukhunjweni kwebhendi okanye ukungonwabi ngokwaneleyo, okukhokelela kumazinga e-HbA anyuswe ngobuxoki.

Ukuthintela ukungcoliseka kwe-cellulose acetate membrane kunye neeprotheni.

Umsinga akufuneki ube phezulu kakhulu; kungenjalo, iibhandi ze<em>hemoglobin azinakuhlukana.

Soloko ubandakanya iisampulu zabantu abaqhelekileyo kunye neehemoglobins ezingaqhelekanga ezaziwayo njengolawulo.

I-Beijing Liuyi Biotechnology yenza itanki yobuchule ye-electrophoresis ye-hemoglobin electrophoresis eyimodeliI-DYCP-38CI-cellulose acetate inwebu ye-electrophoresis itanki, kwaye kukho iimodeli ezimbini zobonelelo lwamandla e-electrophoresis ezikhoyo kwi-cellulose acetate membrane electrophoresis tank.I-DYY-2CkwayeDYY-6Cukunikezwa Amandla.

Ngeli xesha, iBeijing Liuyi Biotechnology ibonelela nge-cellulose acetate membrane kubathengi, kwaye ubungakanani be-cellulose acetate membrane bunokwenziwa ngokwezifiso. Wamkelekile ukuba usibuze iisampuli kunye nolwazi oluthe kratya.

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Ngoku sikhangela amaqabane, zombini itanki ye-OEM electrophoresis kunye nabasasazi bamkelwe.

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