Experiment Principle
Hemoglobin electrophoresis ine chinangwa chekuona uye kusimbisa akasiyana siyana akajairwa uye asina kujairika hemoglobins.
Nekuda kwekuchaja kwakasiyana uye isoelectric mapoinzi emhando dzakasiyana dzehemoglobin, mune imwe pH buffer mhinduro, kana isoelectric point yehemoglobin yakadzikira pane pH yebuffer solution, hemoglobin inotakura zvakaipa uye inotamira kune anode panguva ye electrophoresis. Sezvineiwo, hemoglobin ine chaji yakanaka inofamba ichienda kune cathode.
Pasi peimwe voltage uye mushure menguva yakatarwa ye electrophoresis, hemoglobins ine machaji akasiyana uye uremu hwemamorekuru anoratidza nzira dzakasiyana dzekutama uye kumhanya. Izvi zvinobvumira kupatsanurwa kwenzvimbo dzakasiyana, uye inotevera colorimetric kana electrophoretic scanning ongororo inogona kuitwa munzvimbo idzi kuenzanisa hemoglobins dzakasiyana. Nzira inonyanya kushandiswa ndeye pH 8.6 cellulose acetate membrane electrophoresis.
Mukati me cytoplasm, mapoka ethylene glycol (CHOH-CHOH) aripo mu glycogen kana polysaccharide zvinhu (zvakadai semucopolysaccharides, mucoproteins, glycoproteins, glycolipids, etc.) inonzi oxidized ne periodic acid uye inoshandurwa kuva aldehyde mapoka (CHO-CHO). Aya mapoka ealdehyde anosanganiswa neiyo isina ruvara yepurplish-tsvuku Schiff reagent, achigadzira dhayi-tsvuku-tsvuku inoisa uko mapolysaccharides aripo muchitokisi. Kuita uku kunozivikanwa se periodic acid-Schiff (PAS) staining, yaimbonzi glycogen staining.
Experiment Method
Zvekushandisa:Cellulose acetatemembrane, electrophoresis apparatus(DYCP-38C uye magetsi DYY-6C), Superior Sample Loading Tool (pipette), spectrophotometer, colorimetric cuvettes, buffers.
Buffer:
(1) pH 8.6 TEB Buffer: Wema 10.29 g Tris, 0.6 g EDTA, 3.2 g boric acid, uye wedzera mvura yakasvibiswa kusvika ku1000 ml.
(2) Borate Buffer: Weresa 6.87 g borax uye 5.56 g boric acid, uye wedzera mvura yakasvibiswa kusvika ku1000 ml.
Maitiro:
PKugadziriswa kweHemoglobin Solution
Tora 3 ml yeropa rine heparin kana sodium citrate se anticoagulant. Centrifuge pa2000 rpm kwemaminitsi gumi uye urase plasma. Geza masero matsvuku eropa katatu nesaline yemuviri (750 rpm, 5 maminetsi centrifugation nguva imwe neimwe). Centrifuge pa2200 rpm kwemaminetsi gumi uye bvisa iyo supernatant. Wedzera huwandu hwakaenzana hwemvura yakasvibiswa, wobva wawedzera 0.5 times volume ye carbon tetrachloride. Kuzunungusa nesimba kwemaminitsi mashanu, uye ipapo centrifuge pa 2200 rpm kwemaminetsi gumi kuunganidza iyo yepamusoro Hb mhinduro yekushandisa gare gare.
Kunyorova Membrane
Cheka iyo cellulose acetate membrane kuita mitsetse inoyera 3 cm × 8 cm. Inyure mupH 8.6 TEB buffer kusvika yazara, wobvisa wopukuta nefirita bepa.
Spotting
Shandisa pipette kuona 10 μl yehemoglobin solution yakatwasuka pacellulose acetate membrane (yakashata side), ingangoita 1.5 cm kubva kumucheto.
Electrophoresis
Dururirai borate buffer solution mukamuri ye electrophoresis. Isa iyo cellulose acetate membrane nedivi rine mavara pamucheto wecathode wekamuri. Mhanya pa 200 V kwemaminitsi makumi matatu.
Elution
Cheka nzvimbo dzeHbA neHbA2, wodziisa mumachubhu ebvunzo akasiyana, wowedzera 15 ml uye 3 ml yemvura yakadirwa, zvichiteerana. Zvinyoro zunza kuti ubvise hemoglobin zvachose, wobva wasanganisa.
Colorimetry
Zero kunyura uchishandisa mvura yakanyungudutswa kune elution mhinduro uye kuyera kunyura pa415 nm.
Calculation
HbA2(%) = Kushaikwa kweHbA2 chubhu / (Kubviswa kweHbA chubhu × 5 + Kubvisa HbA2 chubhu) × 100%
Experimental Results Calculation
Reference Range ye pH 8.6 TEB Buffer Cellulose Acetate Electrophoresis: HbA > 95%, HbA2 1% -3.1%
Notes
Electrophoresis nguva haifaniri kuva yakareba. Iyo cellulose acetate membrane haifanirwe kuoma panguva ye electrophoresis. Misa electrophoresis apo HbA neHbA2 zvakanyatsoparadzaniswa. Kurebesa electrophoresis kunogona kukonzera kupararira kwebhendi uye kudzima.
Dzivisa kushandisa sampuli yakawandisa. Yakawandisa hemoglobin fluid inogona kutungamirira kune bhendi detachment kana kusakwana kusvibiswa, zvichiita kuti mazinga eHbA awedzere.
Dzivirira kusvibiswa kwe cellulose acetate membrane ine mapuroteni.
Ikozvino haifaniri kunge yakanyanyisa; kana zvisina kudaro, mabhendi ehemoglobin anogona kusaparadzana.
Gara uchisanganisira zvienzaniso kubva kuvanhuwo zvavo uye zvinodiwa zvinozivikanwa hemoglobins zvisina kujairika sekutonga.
Beijing Liuyi Biotechnology inogadzira tangi ye electrophoresis ye hemoglobin electrophoresis inova modhi.DYCP-38Ccellulose acetate membrane electrophoresis tank, uye kune mhando mbiri dze electrophoresis simba rekupa rinowanikwa kune cellulose acetate membrane electrophoresis tank.DYY-2CuyeDYY-6Cmagetsi.
Zvichakadaro, Beijing Liuyi Biotechnology inopa cellulose acetate membrane kune vatengi, uye saizi ye cellulose acetate membrane inogona kugadzirwa. Kugamuchirwa kuti utibvunze sampuli uye rumwe ruzivo.
Beijing Liuyi mhando ine makore anopfuura makumi mashanu nhoroondo muChina uye kambani inogona kupa yakagadzikana uye yemhando yepamusoro zvigadzirwa pasi rese. Kupfuura makore ebudiriro, yakakodzera sarudzo yako!
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Nguva yekutumira: Sep-20-2023