· i-polyacrylamideijeli kufanele nge-acrylamide monomer, i-polymerization yokuqala impahla, i-catalyst, kanye nelungelo likasawoti kanye nengxube ye-buffer ndawonye.
· i-acrylamidekanye ne-BIS (N, N '- methylene double acrylamide) i-monomer form gel matrix.
I-ammonium persulfate iqala inqubo ye-adhesive polymerization.Ukwakhiwa kweglue kudinga isisombululo se-ammonium persulfate esingu-10% esilungiselelwe emanzini.Iningi lolwazi libonise isidingo sokusebenzisa okusebenzayo.Nokho, isixazululo esingu-10% singabekwa ku-4℃ amasonto ambalwa ngaphandle kokulahlekelwa okukhulu komsebenzi.Yenza kufike ku-10 ml bese ulahla lapho iglue yehluleka ukuhlanganisa.
Ithiphu: Iphesenti lei-acrylamideekulandeleni iglue ne-protein glue akufani.Uma usebenzisa i-premade acrylamide: Isixazululo se-BIS, qiniseka ukuthi uthola ibhodlela elifanele.
· I-TEMED (N, N, N ', N '- tetramethyl ethylenediamine) iyi-catalyst, ebhodleleni elinsundu, elibekwe esiqandisini.Engeza ngaphambi nje kokuthela i-glue.
Ingilazi ye-polyacrylamide electrophoresis esetshenziswe ku-electrophoresis kufanele igezwe ngaphambi nangemva kwesikhathi ngasinye.Ngemuva kwe-electrophoresis, geza ngebhulashi elithambile nendwangu emanzini ashisayo anensipho, hlambulula ngamanzi a-distilled bese ume ukuze wome.
· Umswakama kanye nothuli kungabangela nge-polymer engenalutho.Ngaphambi kwe-electrophoresis, hlanza ipuleti lengilazi ngesicoci sengilazi bese usula ngebhulashi elithambile.Geza ngamanzi a-distilled bese usula kahle ngephepha lokusula.Ukugeza nge-ethanol engu-70% ngaphambi kokusula ngephepha kusiza ukuhlanza nokusheshisa ukomisa.Engeza amasampula e-acrylamide ngokulandelana: I-BIS, amanzi, isisombululo se-buffer, i-ammonium persulfate, i-TEMED.Shake kahle futhi uthele ngokushesha.
Akudingekile ukususa i-polyacrylamide ngaphambi kwe-polymerization.(I-Acrylamide yayivamise ukufakwa ku-vacuum ukuze kukhishwe amabhamuza ngenxa yokuthi umoya-mpilo uvimbela ukwenziwa kwe-polymerization.)
Amathiphu esampula ephoyinti leglue elivundlile.
faka ucezu lwephepha elimnyama ebhokisini elingezansi, ingemuva elimnyama ukuze wenze isampula yembobo ukuze ubone ngokucacile.
· I-glue ithangi eligcwele isigcinalwazi, ngaphezu nje kwe-colloid.
· uma unqenqema lunokukhanya, khanyisa izibani, vumela ukukhanya kukhanye i-colloid.Dweba isampula ku-pipette.
· usebenzisa idivayisi yokufaka ipayipi ezenzakalelayo.
· ku-10-200 mu 1 uketshezi olunyakazayo lungasetshenziswa kumaphoyinti amaningi kusampula.Ngezimbobo zesampula ezincane kakhulu (ngaphansi kuka-10μ1), ikhanda elide le-pipette elisetshenziselwa ukulandelana kweglue lilula kakhulu.
· ukufakwa kwamapayipi kusanda kungena kusampula, ukuhogela uketshezi oluhamba kancane.Isampula ingase ibonakale inamathele ne-glycerin, futhi ukupompa okusheshayo kungase kudonsele amabhamuza omoya ekhanda le-pipette.
· amasampula ngemva kokuhogela ikhanda lamapayipi, azohambisa ikhanda eliwuketshezi kancane onqenqemeni lwepayipi noma iphepha lokusula amunca ikhanda eliwuketshezi ngaphandle kwamaconsi.Qaphela ukuthi ungamunyi isampula.
Faka isampula emgodini wesampula
· I-pipetting device ukugcina ingcindezi encane, yenza amasampula anyakaze kancane ekhanda eliwuketshezi elichichimayo.
· ikhanda elibhobhozayo elifakwe ku-buffer, liphakeme kancane kunezimbobo zamabala, gcina ingcindezi eqondile.Iphuzu le-pipette lingafakwa emgodini omncane.
· kancane futhi kancane amasampula akhishwe.Ithiphu ye-pipette ibekwe ngaphezu kwembobo yesampula yephuzu, futhi isampula izocwila emgodini.Vumela isampula lizike ukugcwalisa imbobo yesampula esikhundleni sokuphusha phakathi.
· uma ithonsi lokugcina lesampula elinekhanda eliwuketshezi, uketshezi luzoya emlenzeni wesibili, luphakamise kancane ukunyakaza koketshezi, lusuke ku-buffer.
Indlela yokwenza isampula yeglue eqondile?
· Izimbobo zesampula eziqondile zeglue ezakhiwe phakathi kwezingcezu ezimbili zengilazi.Ngeglue elincanyana kakhulu, ikhanda le-pipette alikwazi ngisho nokufakwa phakathi kwamapuleti amabili engilazi.Buka i-glycerin!Beka ikhanda le-pipette phezu kwembobo yesampula futhi isampula izocwila emgodini.
· Isampula lephuzu ngaphambili, qiniseka ukuthi ubeka iphuzu elimile le-polypropylene acyl gel imbobo yesampula iyahashwa ihlanzeke.Geza i-acrylamide enganqunywanga kanye namanzi angase avele ngaphansi kwembobo yesampula.Amanzi angenza imbobo yesampula ibe mncane kakhulu.Sebenzisa isirinji engu-25ml noma engu-50ml kanye nenaliti yegeji engu-18.Thela i-electrophoresis buffer bese usula ngokucophelela imbobo yesampula.
· Kungaba nzima ukubona imbobo yesampula, kodwa ngesikhathi esifanayo, okunye kulula.Uma kunezimbobo ezeqile, ibhafa yesampula ingahlolwa nge-bromophenol eluhlaza okwesibhakabhaka.
Isikhathi sokuthumela: Mar-31-2023