Izindlela zokuphepha zePolyyamide Gel Electrophoresis

· polycrylamideIjeli kumele nge-acrylamide monomer, i-polymerization iqala ngokwezinto ezibonakalayo, i-catalyst, kanye nelungelo likasawoti kanye nengxube yengxube ye-buffer ndawonye.

· acrylangaideFuthi i-BIS (N, N '- Methylene Double AcRlamide) yi-monomer form gel matrix.

· Ammonium Perselfate Qala i-Adhesive Polymerization Process. Ukwakhiwa kwe-glue kudinga isixazululo esingu-10% Ammonium Perselfate Solution esilungiselelwe emanzini. Iningi lemininingwane ikhombise isidingo sokusebenzisa okusebenzayo. Kodwa-ke, isixazululo esingu-10% singabekwa ku-4 ② ② amasonto ambalwa ngaphandle kokulahleka okukhulu komsebenzi. Yenza kufike ku-10 ml bese ulahla lapho i-glue yehluleka ukuhlanganisa.

ICEBISO: Iphesenti leacrylangaideNgokulandelana kwe-glue ne-glue protein akufani. Uma usebenzisa i-Prepade Acrymide: Isixazululo se-BIS, qiniseka ukuthola ibhodlela elifanele.

· Ithempeli (n, n, n ', n' - tetramethyl ethylediamine) iyi-catalyst, ebhodleleni elinsundu, elifakwe esiqandisini. Engeza ngaphambi nje kokuthulula glue.

· Polycryrylamide electoferesis ingilazi esetshenzisiwe ku-electrophoresis kufanele igezwe ngaphambi nangemva kwesikhathi ngasinye. Ngemuva kwe-electrophoresis, geza ngebhulashi elithambile nendwangu emanzini ashisayo anensipho, hlambulula ngamanzi anciphile bese ume ukuze womile.

· Umswakama nothuli kungadala nge-polymer eyize. Ngaphambi kwe-electrophoresis, hlanza ipuleti lengilazi nge-ligs cleaner bese ulisusa ngebhulashi elithambile. Geza ngamanzi anciphile bese womile kahle ngephepha Sula. Ukuhlanza nge-70% ethanol ngaphambi kokusula ngephepha kusiza ekuhlanzeni nasekusheshiseni ukoma. Ngeza amasampula we-acrylamide ngokulandelana: i-BIS, Amanzi, Isixazululo Se-Buffer, i-ammonium Perselfate, Ithempeli. Shake kahle bese uthele ngokushesha.

Akudingekile ku-degass polylacrylamide ngaphambi kwe-polymerization. (I-Acrylamide esetshenziselwe ukubekwa ecaleni ukususa ama-bubble ngoba umoya-mpilo uvimbela i-polymerization.)

Izeluleko ze-Glue Point Point Tamputer.

· Beka ucezu lwephepha elimnyama ebhokisini elingezansi, ingemuva elimnyama ukwenza umgodi oyisampula ukuze ubone ngokucacile.
· Twenk tank buffer, ngaphezulu kwe-colloid.
· Uma umphetho unesibani, vula amalambu, vumela ukukhanya kukhanya colloid. Dweba isampula ku-pipette.
Sebenzisa idivaysi ye-othomathikhi ye-pipetting.
Iphuzu le-10-200 Musite elihambayo lingasetshenziswa emaphoyinti amaningi kwisampula. Ngemigodi emincane kakhulu yesampula (ngaphansi kuka-10μ1), ikhanda elide le-pipette elisetshenziselwa ukulandelana kwe-glue lilula.

· Pipetting usanda kucwiliswa kwisampula, ephefumula kancane kancane i-fluid. Isampula ingahle ibonakale inamathele nge-glycerin, futhi ukupompa okusheshayo kungadonsa ama-bubble omoya ekhanda le-pipette.

· Samasampula ngemuva kokuphefumula ikhanda, uzohambisa ikhanda lamanzi ngobumnene emaphethelweni epayipi noma iphepha lokusula lincela ikhanda eliwuketshezi ngaphandle kwamaconsi. Qikelela ukuthi ungamunca isampula.

Faka isampula emgodini wesampula

· Idivaysi yokugcina ingcindezi encane, yenza amasampula ahambisane kancane ngekhanda eliwuketshezi.
Ikhanda le-Pipetting lifakwe kwi-buffer, liphakeme kancane kunezimbobo zendawo, gcina ingcindezi eyakhayo. Ithiphu ye-pipette ingafakwa emgodini omncane.
· Kancane futhi ngokuqinile amasampula ngaphandle. Ithiphu ye-Pipette ibekwe ngaphezulu komgodi wesampula wePhoyinti, futhi isampula lizocwila emgodini. Vumela ukucwila kwesampula ukugcwalisa umgodi oyisampula esikhundleni sokucindezela ngaphakathi.
· Lapho ethonsi lokugcina lesampula elinekhanda eliwuketshezi, uketshezi luzodlulela emlenzeni wesibili, luphakamise kancane kancane ukunyakaza koketshezi, phuma ku-buffer

Ungenza kanjani amasampula we-glue?

· Vertical glue ukhomba izimbobo zesampula zakhiwa phakathi kwezingcezu ezimbili zengilazi. Ku-glue emincane kakhulu, ikhanda le-pipette alikwazi ngisho nokufakwa phakathi kwamapuleti amabili engilazi. Bukela i-glycerin! Beka ikhanda le-pipette phezu komgodi wesampula bese isampula lizocwila emgodini.
Isampula ye-Point ngaphambili, qiniseka ukubeka iphuzu mpo ye-polypropylene acyl gel gel hole hole hole ihlanzekile. Geza i-acrylamide engabekezeleleki namanzi angavela ezansi komgodi wesampula. Amanzi angenza umgodi oyisampula mncane kakhulu. Sebenzisa isirinji engu-25ml noma i-50ML nenaliti engu-18-geuge. Thela ku-Olectrophoresis Buffer bese ucindezela ngokucophelela umgodi oyisampula.
Kungaba nzima ukubona umgodi oyisampula, kepha ngokufanayo, okusele kulula. Uma kunezimbobo eziningi ngokweqile, i-buffer yesampula ingahlolwa nge-bromophenol eluhlaza okwesibhakabhaka.