Sehlooho: Mekhoa ea Electrophoresis ho Biochemistry
Selelekela
Electrophoresis ke mokhoa oa bohlokoa oa biochemistry le baeloji ea limolek'hule, oa bohlokoa haholo bakeng sa tlhahlobo le karohano ea limolek'hule tse kholo, tse kang li-nucleic acid le liprotheine. Mokhoa ona o sebelisa ho falla ho fapaneng ha likaroloana tse nang le charged tlas'a tšimo ea motlakase, o nolofalletsa bo-rasaense ho arola metsoako e rarahaneng likarolong tsa bona ka bomong. Electrophoresis ha e fane feela ka temohisiso e tebileng mabapi le libaka tsa sebopeho le ts'ebetso ea limolek'hule empa hape e bohlokoa ho tlhahlobo, forensics le lits'ebetsong tse ngata tsa lipatlisiso. Sengoloa sena se hlahloba mekhoa e fapaneng ea electrophoresis ho biochemistry, se totobatsa melao-motheo ea eona, lits'ebetso le tsoelo-pele.
Melao-motheo ea Electrophoresis
Motheong oa eona, electrophoresis e sebetsa ho latela molao-motheo oa hore limolek'hule tse chajileng lia falla ha li beoa tšimong ea motlakase. Sekhahla sa ho falla se susumetsoa ke lintlha tse 'maloa: tjhaja ea molek'hule, boholo le sebopeho sa molek'hule, matla a tšimo ea motlakase, le litšobotsi tsa sepakapaka seo limolek'hule li tsamaeang ho sona. Ka kakaretso, sesebelisoa sa electrophoresis se na le phepelo ea motlakase, li-electrode tse peli, le matrix ea gel e sebetsang e le sepakapaka se tšehetsang.
Jel, e entsoeng ka agarose kapa polyacrylamide, e sebetsa e le sefe ea limolek'hule. Likaroloana tse nyane kapa tse nang le tjhaja e ngata li tla tsamaea kapele ka har'a masoba a jel, ha likaroloana tse kholo kapa tse nang le tjhaja e nyane li tsamaea butle haholo. Ka ho bapisa motsamao oa lisampole tsa liteko le maemo kapa matšoao a tsejoang, bafuputsi ba ka tseba le ho hlalosa likarolo tse ka har'a sampole.
Mefuta ea Mekhoa ea Electrophoresis
1. Agarose Gel Electrophoresis (LILEMO)
Hangata electrophoresis ea gel ea Agarose e sebelisoa bakeng sa karohano ea likaroloana tsa nucleic acid. Matrix ea gel ea agarose e lumella qeto ea limolek'hule tsa DNA kapa RNA ho tloha ho lipara tse makholo a 'maloa tsa motheo ho isa ho li-kilobase tse' maloa. Kamora ho kenya sampole ka har'a liliba, motlakase oa sebelisoa, o etsang hore li-nucleic acid li fallele ho anode ka lebaka la tjhaja ea tsona e mpe. Sephetho se bonoa ka ho sebelisa lidae tse kopanyang tse kang ethidium bromide tlas'a khanya ea UV, e lumellang ho lekanya le ho sekaseka bohloeki le boholo ba nucleic acid.
2. Polyacrylamide Gel Electrophoresis (LEQEPHE)
Le hoja agarose gel electrophoresis e loketse li-nucleic acid tse kholoanyane, polyacrylamide gel electrophoresis (PAGE) e loketse ho rarolla likaroloana tse nyane tsa DNA, RNA le liprotheine. PAGE e fana ka qeto e phahameng ka lebaka la taolo e nyane holim'a boholo ba pore ea gel, e fetoloang ka ho fetola mahloriso a acrylamide. SDS-PAGE, mofuta oa mokhoa ona, e sebelisoa ka ho khetheha bakeng sa karohano ea protheine. Liprotheine lia qhekelloa 'me li koahetsoe ka sodium dodecyl sulfate (SDS), e leng se li fang karolelano e mpe ea charge-to-mass. Sena se tiisa hore karohano nakong ea electrophoresis e thehiloe haholo-holo boima ba limolek'hule.
3. Capillary Electrophoresis (CE)
Capillary electrophoresis ke mokhoa o nang le qeto e phahameng o bohlokoa bakeng sa ho sekaseka limolek'hule tse nyane ka katleho le lebelo le leholo ho feta gel electrophoresis ea setso. Ho CE, lisampole li kenngoa ka har'a tube ea capillary e tletseng tharollo ea electrolyte. Tšimo ea motlakase e sebelisoa, e leng se etsang hore likarolo tse fapaneng li falle ka lebelo le fapaneng ho latela likarolelano tsa tsona tsa charge-to-mass. Sekala se senyenyane sa capillary se fokotsa tlhahiso ea mocheso le litlamorao tsa ho hasana haholo, e leng se fellang ka qeto e matla. Capillary electrophoresis e ka etsoa ka mekhoa e kang capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC), le tse ling.
4. Ho Tsepamisa Motlakase (IEF)
Ho tsepamisa maikutlo ka isoelectric ke mokhoa o matla oa ho arola liprotheine ho latela lintlha tsa tsona tsa isoelectric (pI). Mokhoa ona o kenyelletsa ts'ebeliso ea tšimo ea motlakase holim'a gel e nang le pH gradient. Liprotheine li tsamaea ka har'a gel ho fihlela li fihla ntlheng eo pH e lekanang le pI ea tsona, eo ho eona li se nang tefiso ea net 'me kahoo li lula li sa sisinyehe. Mokhoa ona o lumella karohano ea liprotheine ka qeto e phahameng, 'me e ka kopanngoa le SDS-PAGE ka electrophoresis ea mahlakore a mabeli (2D-PAGE) bakeng sa tlhahlobo e ntlafalitsoeng le ho feta.
5. Electrophoresis ea Gel ea Tšimo e Pulsed (PFGE)
Ho sebetsana le mefokolo ea gel electrophoresis ea setso ho aroleng limolek'hule tse kholo haholo tsa DNA (tse fetang 20 kb), pulsed-field gel electrophoresis e sebelisa masimo a motlakase a fapanyetsanang ho tsamaea litseleng tse rarahaneng tsa ho falla ha likaroloana tse kholo tsa DNA ka har'a matrix ea gel. PFGE e bohlokoa haholo lithutong tsa genotyping le genome mapping, e fanang ka temohisiso e qaqileng ka meaho ea chromosome le likarolo tse kholo tsa lefutso.
dikopo
Mekhoa ea electrophoresis e sebelisoa haholo mafapheng a fapaneng a saense:
1. Ho Klonngoa ha Dimolekhule le Genomics: Ho arolwa le ho hlahlojwa ha dikotwana tsa DNA kamora thibelo ya tshilo ya dijo kapa ho atoloswa ha PCR.
2. Proteomics: Tlhaloso le kgethollo ya diprotheine, ho kenyeletswa le diphetoho tsa kamora phetolelo le dikamano tsa protheine le protheine.
3. Tlhahlobo ea Bongaka: Ho tsebahatsa liphetoho tsa lefutso, lintho tse tšoaetsanoang, le li-biomarker.
4. Forensic: Ho nka menwana ya DNA le ho etsa dipatlisiso tsa ditlolo tsa molao.
5. Taolo ea Boleng ho Biotechnology: Ho netefatsa bohloeki le boleng ba lihlahisoa tsa biotechnology tse kang liente, li-enzyme le li-antibodies.
Tsoelo-pele le Litaelo tsa Kamoso
Sebaka sa electrophoresis se ntse se fetoha ka mehla, ka kgatelopele e kgolo e ntlafatsang qeto, lebelo le kutlwisiso. Dintho tse ntjha tse kang electrophoresis ya microfluidic di kopanya miniaturization le mekgwa ya electrophoretic, e betla tsela bakeng sa disebediswa tsa laboratori tse fanang ka bokgoni ba ho hlahloba ka potlako, ba tlhokomelo. Mekhoa e ntlafaditsweng ya ho lemoha, ho kenyeletswa le fluorescence le mass spectrometry coupling, e atolosa meedi ya seo se ka fihlellwang ka karohano ya electrophoretic.
Ho feta moo, mefuta ea electrophoresis ea othomathiki le e nang le matla a holimo e khotsofatsa litlhoko tse ntseng li hola tsa lithuto tse kholo tsa genomic le proteomic, moo ho sebetsana le palo e kholo ea lisampole ka potlako le ka nepo ho leng bohlokoa.
fihlela qeto e
Electrophoresis e ntse e le sesebelisoa sa bohlokoa le se matla ho biochemistry, e ntse e ikamahanya le maemo khafetsa ho tobana le liphephetso tsa tšimo e tsoelang pele ea baeloji ea limolek'hule. Ka ho utloisisa le ho sebelisa melao-motheo le mekhoa ea electrophoresis, bo-rasaense ba ka fihlela litlhahlobo tse nepahetseng le tse qaqileng tsa limolek'hule tse rarahaneng tsa baeloji, ba khanna boqapi bo ntlafetseng lipatlisisong, tlhahlobong le nts'etsopele ea kalafo. Bokamoso ba electrophoresis bo tšepisa kopanyo le katleho e kholoanyane, ho netefatsa hore e tsoela pele ho phetha karolo ea bohlokoa ho sibolloeng ha saense.
