· polyacrylamideijeli kufuneka nge-acrylamide monoma, i-polymerization yokuqala imathiriyeli, i-catalyst, kunye nelungelo letyuwa kunye nomxube we-buffer kunye.
· acrylamidekunye ne-BIS (N, N '- methylene double acrylamide) yimonomer form gel matrix.
· ammonium persulfate qala inkqubo yokuncamathelisa polymerization.Ukuqulunqwa kweglue kufuna i-10% yesisombululo se-ammonium persulfate esilungiselelwe emanzini.Uninzi lolwazi lubonise imfuno yokusetyenziswa ngokusebenzayo.Nangona kunjalo, isisombululo se-10% sinokubekwa kwi-4 ℃ iiveki ezininzi ngaphandle kokulahlekelwa okukhulu komsebenzi.Yenza ukuya kwi-10 ml kwaye ulahle xa iglue ihluleka ukudibanisa.
Ingcebiso: Ipesenti yeacrylamideekulandeleni iglue kunye neprotein glue ayifani.Ukuba usebenzisa i-acrylamide yangaphambili: isisombululo se-BIS, qiniseka ukuba ufumana ibhotile efanelekileyo.
· I-TEMED (N, N, N ', N '- tetramethyl ethylenediamine) i-catalyst, kwibhotile emdaka, efakwe efrijini.Yongeza ngaphambi nje kokugalela iglue.
· Polyacrylamide electrophoresis esetyenziswa iglasi electrophoresis kufuneka ihlanjwe phambi nasemva kwexesha ngalinye.Emva kwe-electrophoresis, hlamba ngebrashi ethambileyo kunye nelaphu emanzini ashushu anesepha, hlambulula ngamanzi adibeneyo kwaye ume ukuze wome.
· Ukufuma kunye nothuli kunokubangela ngepolymer engenanto.Ngaphambi kwe-electrophoresis, coca ipleyiti yeglasi ngesicoci seglasi kwaye uyisule ngebrashi ethambileyo.Geza ngamanzi adibeneyo kwaye uyomise ngokucokisekileyo ngephepha lokusula.Ukuhlanjululwa nge-ethanol engama-70% ngaphambi kokusula ngephepha kunceda ukucoca nokukhawulezisa ukomisa.Yongeza iisampuli ze-acrylamide ngokulandelelanayo: i-BIS, amanzi, isisombululo se-buffer, i-ammonium persulfate, i-TEMED.Gcoba kakuhle kwaye uthele ngokukhawuleza.
Akuyomfuneko ukuba degass polyacrylamide phambi polymerization.(I-Acrylamide yayidla ngokufakwa kwivacuum ukususa amaqamza kuba ioksijini inqanda ukwenziwa kwepolymerization.)
Iingcebiso zeesampuli zeglue ezithe tye.
beka isiqwenga sephepha elimnyama kwibhokisi esezantsi, imvelaphi emnyama ukwenza isampulu yomngxuma ukubona ngokucacileyo.
· Itanki yeglu egcwele isithinteli, ngaphaya kwecolloid.
· ukuba umda unokukhanya, vula izibane, ukukhanya makukhanye i-colloid.Zoba isampuli kwipipette.
· usebenzisa isixhobo sokuzifakela umbhobho.
· kwi-10-200 mu 1 indawo yolwelo ehambayo ingasetyenziswa kuninzi lwamanqaku kwisampulu.Kwimingxuma yesampulu encinci kakhulu (ngaphantsi kwe-10μ1), intloko ende yepayipi esetyenziselwa ukulandelelanisa iglu ilungele ngakumbi.
· ukuntywiliselwa nje ngemibhobho kwisampulu, ukusezela ulwelo oluhamba kancinane.Isampuli ingabonakala inamathele kunye ne-glycerin, kwaye ukupompa ngokukhawuleza kunokutsala ama-bubbles emoyeni kwintloko ye-pipette.
• iisampulu emva kokuphefumlela intloko yombhobho, ziyakuhambisa intloko yolwelo ngobunono kwincam yombhobho okanye izosula iphepha lokufunxa intloko engamanzi ngaphandle kwamathontsi.Qaphela ukuba ungafunxi isampuli.
Beka isampuli kumngxuma wesampuli
· isixhobo sombhobho ukugcina uxinzelelo oluncinci, yenza iisampuli zishukumise kancinane intloko yolwelo ephuphumayo.
· Intloko yombhobho efakwe kwisithinteli, iphakame kancinci kunemingxuma yamabala, gcina uxinzelelo olulungileyo.Incam yepipette ingafakwa kumngxuma omncinci.
· ngokucothayo nangokucothayo iisampuli zikhutshwe.Iphuzu le-pipette libekwe ngaphezu komngxuma wesampuli yenqaku, kwaye isampuli iya kutshona emngxunyeni.Vumela isampulu itshone igcwalise umngxuma wesampulu endaweni yokutyhala ngaphakathi.
· Kanye ithontsi lokugqibela lesampulu enentloko elulwelo, ulwelo luza kuya kumlenze wesibini, ngokucothayo lunyuse intshukumo yolwelo, luphume kwisithinteli.
Ungayenza njani isampuli yeglue ethe nkqo?
• imingxunya yesampulu yeglu ethe nkqo eyenziwe phakathi kwamaqhekeza amabini eglasi.Kwiglue encinci kakhulu, intloko ye-pipette ayikwazi ukufakwa phakathi kweeplate ezimbini zeglasi.Jonga i-glycerin!Beka intloko ye-pipette phezu komngxuma wesampuli kwaye isampuli iya kutshona emngxunyeni.
• isampuli yenqaku ngaphambili, qiniseka ukuba ubeka inqaku elithe nkqo le-polypropylene acyl gel isampulu yomngxuma ihlanjululwe icocekile.Hlamba i-acrylamide engaxutywanga kunye namanzi anokubonakala ezantsi kumngxuma wesampulu.Amanzi anokwenza umngxuma wesampulu ube mncinci kakhulu.Sebenzisa i-25ml okanye i-50ml yesirinji kunye nenaliti ye-18-gauge.Galela kwi-electrophoresis buffer kwaye ugungxule ngononophelo umngxuma wesampulu.
· Kunokuba nzima ukubona umngxuma wesampulu, kodwa kwangaxeshanye, okunye kulula.Ukuba kukho imingxunya egqithisileyo, isampulu yesithinteli sinokuvavanywa ngebromophenol eluhlaza okwesibhakabhaka.
Ixesha lokuposa: Mar-31-2023