Ma `lumot

GST yorlig'i bilan oqsilni tozalash paytida ustunni qanday yuvish mumkin?


Men so'nggi 4 yil davomida GST bilan belgilangan oqsillar bilan ishladim va hujayra lizatini ustunga yuklaganimdan so'ng, men uni 20-30 kolonnali hajmli PBS bilan yuvdim va ba'zida mening oqsillarim juda toza, ba'zan esa juda ko'p aralashmalar bilan tozalandi.

Bir necha marta NaCl gradienti (0-400 mM) bilan yuvishga harakat qildim, lekin keyinchalik buni qilmaslikka qaror qildim, chunki u oqsilni denatüratsiya qilishi va ba'zi oqsillarim qayta katlanmasligi mumkin.

Yuvish bosqichi uchun nimani taklif qilasiz?

Eslatma: Menda yuvish/oqim/elutsiyalarni ko'rish uchun hech qanday UV detektorim yo'q.


GST ni glutation agaroz boncuklari bilan tozalashda standart yuvish uchun STE buferidan (10 mM Tris-HCl (pH 7-7,5), 150 mM NaCl va 1 mM EDTA) foydalanish hisoblanadi va biz laboratoriyamizda bu bilan hech qanday muammoga duch kelmadik!


GST-oqsilni tozalash: parchalanish mahsuloti - (14.04.2007)

Mening ovozim pwnererrning tizimingizdagi ikkita plazmid taklifiga mos keladi. Ba'zi hujayralarni o'stiring va plazmid tayyorlang. Agar aralash bo'lsa, darhol ko'rasiz.
BTW, maqsadli oqsil va ifloslantiruvchi moddalarning qancha qismini olasiz? Kontaminant GSH boncuklariga bog'lanadimi? Agar yo'q bo'lsa, yaxshilab yuvib tashlang. Agar u yopishib qolsa, qoldiring. Maqsadli proteinni chiqarish uchun termoyadroviyni hazm qilganingizda, ifloslantiruvchi ustunda qoladi. Agar u chiqib ketgan bo'lsa ham, siz uni o'lcham ustuni bilan olib tashlashingiz mumkin.

Siz boshida ko'p turdagi induktsiya va o'sishni sinab ko'rishingiz shart emas. Odatdagidek 1 ml bakteriyalaringizni ko'paytiring va qo'zg'ating va kutilgan barcha bosqichlarni bajaring. Qaysi bosqichda nima bo'lganini ko'rish uchun jel va ko'k rangni ishlating. Keyin mumkin bo'lgan muammo va echimni ko'rib chiqasiz. Bizni yangilab turingmi? ^___^

Mening oqsilim hujayralarda parchalana boshlaydi degan fikr uchun rahmat. Bu shunday bo'lganga o'xshaydi, men bug, lizat, oqimning GST-immuno-bo'yoq namunalaridan keyin o'zimdan pastda bir nechta chiziqlarni ko'raman. Shunday qilib, endi men o'sish paytida buzilishni minimallashtirishga harakat qilaman. Har qanday g'oyalar qabul qilinadi.

Men oldin nima qilganman:
boshlang'ich madaniyati bilan ommaviy axborot vositalarini 1:10 inokulatsiya qiling
uni OD600 gacha o'stiring: 0,6-1,2 37C da silkitib
IPTG 1mM bilan induktsiya qiling
RTni bir kechada o'stiring

MagicMedia bilan induksiyaga ehtiyoj yo'q, shuning uchun emlashdan keyin men uni RT 24 soat davomida silkitib o'sishiga ruxsat berdim.

Sizningcha, glyukoza yoki qisqaroq o'sish davri menga yordam berishi mumkinmi?

Ba'zi izohlarga:
Degradatsiya mahsuloti GST musbat va ba'zi hollarda u maqsadli protein kabi kuchli.
Afsuski, o'lchamlar o'rtasidagi farq juda oz (men Centriconni allaqachon sinab ko'rganman) sababli, men uni kesish orqali qutulolmasligim mumkin.
Kontaminant boncuklarni xursandchilik bilan bog'laydi va faqat elutsiya paytida chiqadi. Afsuski, menga bu oqsillarga kerak bo'lgan tajribalar uchun proteinimga birlashtirilgan GST yorlig'i kerak.

Vaqtni qisqartiring mening yordamim. Temp haqida ishonchim komil emas. Ba'zan 37 da atigi 2 soat bajaring, degradatsiya kamroq bo'lishi mumkin, ba'zida bu yordam bermaydi. sinab ko'rishingizdan qo'rqaman. GST oqsili nima uchun, baribir, u juda toza bo'lishi kerakmi? Agar buzilish haqida hech narsa qila olmasangiz (sizda sof klon bor deb o'ylayman) va siz juda toza proteinni xohlasangiz, FPLC kabi biror narsa qilishingiz kerak bo'lishi mumkin.

Fikrlar uchun rahmat, men sinab ko'rishni boshladim. Men 6 soatlik RT o'stirdim, hosil yomon edi, lekin degradatsiya ham kamaydi. Lekin men, albatta, 37C da 2-3 soat harakat qilaman (bu ham qulay bo'lar edi). Men qiladigan boshqa narsa - IPTG kontsentratsiyasi bilan o'ynash va glyukoza qo'shishga harakat qilish. Bu boradagi har qanday izoh qadrlanadi.

Menga sof GST yorlig'i bo'lgan protein kerak, chunki u FRET asosidagi bog'lanish tahlili uchun kerak. Shunday qilib, hech bo'lmaganda maqsadli oqsilning ulushi parchalanishdan ancha yuqori bo'lishi kerak.
Aytgancha, FPLC nima? Bu suyuqlik xromatografiyasining bir turimi? Men HPLC yoki boshqa turdagi xromatografiya haqida o'yladim, lekin menda hech qanday ma'lumot yo'q, qaysi biri 26 va 37 kDa oqsillarni ajratish uchun qo'llanilishi mumkin? Men bu texnikalar bilan tanish emasman.

Qanday bo'lmasin, men turli xil o'sish davrlarini, induksiya va glyukozani sinab ko'raman va agar to'g'ri topsam, protokol bilan qaytib kelaman. Yordamingiz uchun rahmat.

GE Healthcare veb-saytidan izlashingiz kerak bo'lgan aniq ma'lumot. Ularga ko'ra, oqsillarni hajmiga qarab ajratish mumkin. Menimcha, mutaxassisdan so'rash kerak.

IPTGga kelsak, biz odatda 0,1-1mM IPTG dan foydalanamiz. Siz 1 mm dan ortiq foydalanmasligingiz kerak. Menimcha, IPTG miqdorini kamaytirish yordam berishi mumkin. Sizning ekspress induktsiyangiz haqida ishonchim komil emas. Biz uchun biz faqat bitta koloniyani tanlaymiz, 400 ml muhitga to'kib tashlaymiz, keyin oqsilni tozalaymiz. Shunday qilib, jami o'sish bir kun (hujayralarni yig'ish uchun koloniyani tanlagandan beri).

Men ilgari hech qachon glyukoza ishlatmaganman, bunda sizga yordam bera olmayman. Kechirasiz.

IPTG (umumiy konsentratsiya) uchun taxminan 1mM dan foydalaning. Agar siz konsentratsiya bilan o'ynashni xohlasangiz, ehtimol 0,5 dan 5 mm gacha. Glyukoza faqat boshqa qo'shimcha hisoblanadi. Kerak emas. Boshqa parametrni qo'shish ishlarni yanada murakkablashtiradi. Bakteriyalaringizni pastroq haroratda, yaxshiroq katlamada o'stirishga harakat qiling.

Xo'sh, GST yorlig'i, tozalash uchun sizga qatron kerak. Lekin haqiqatan ham yaxshi sifat uchun HPLC yoki FPLC ga o'ting. Menimcha, FPLC yaxshiroq tanlov bo'ladi. Ha, bu suyuqlik xromatografiyasining bir turi. Ammo siz HPLC yoki FPLC ga borishdan oldin juda ko'p miqdordagi kerakli proteinni ifoda eta olishingiz kerak. Hammaga yaxshi!

GE Healthcare veb-saytidan izlashingiz kerak bo'lgan aniq ma'lumot. Ularga ko'ra, oqsillarni hajmiga qarab ajratish mumkin. Menimcha, mutaxassisdan so'rash kerak.

IPTGga kelsak, biz odatda 0,1-1mM IPTG dan foydalanamiz. Siz 1 mm dan ortiq foydalanmasligingiz kerak. Menimcha, IPTG miqdorini kamaytirish yordam berishi mumkin. Sizning ekspress induktsiyangiz haqida ishonchim komil emas. Biz uchun biz faqat bitta koloniyani tanlaymiz, 400 ml muhitga to'kib tashlaymiz, keyin oqsilni tozalaymiz. Shunday qilib, jami o'sish bir kun (hujayralarni yig'ish uchun koloniyani tanlagandan beri).

Men ilgari hech qachon glyukoza ishlatmaganman, bunda sizga yordam bera olmayman. Kechirasiz.

Albatta, ularni o'lchamiga qarab ajratish mumkin. Etarlicha uzun ustun bilan siz har qanday narsani o'lchamiga ko'ra ajratishingiz mumkin. FPLC juda yaxshi, lekin baribir u o'lchamni istisno qilish yomon va har qanday holatda ham undan qochish kerakligi bilan bog'liq emas. Bugungi kunda oqsilni hajmi bo'yicha tozalashning juda yaxshi usullari mavjud. O'lchamni istisno qilishning ko'plab sabablari bor, ular orasida, lekin cheklanmagan holda, bu past piksellar soniga ega va past ixchamlik usuli va, albatta, bu abadiy davom etadi. Ion almashtirgichning bir turiga yopishib oling.

Glyukoza qo'shish yoki IPTG kontsentratsiyasi bilan o'ynashni bezovta qilmang. Bu narsalar sizga yordam bermaydi va vaqtni butunlay behuda sarflashdir. Bu plazmidlarning ifodasi deyarli hammasi yoki hech narsa emas. Ya'ni, 0,1 mM IPTG, ehtimol, 1 mM bilan deyarli bir xil ifoda darajasini beradi. U unchalik titrlash mumkin emas.

Ha, ion almashtirgich yaxshi, lekin bu erda muammo buzilish bilan bog'liq bo'lishi mumkin. Proteinni o'zining parchalanish mahsulotlaridan tozalash uchun ion almashtirgich o'lchamlarni ajratish kabi foydali bo'lmasligi mumkin. Albatta, bu erda turli xil o'lchamlar unchalik yaxshi emasligi sababli (26 va 37kD), u uzoq va sekin bo'ladi, qo'rqaman.

Ha, ion almashtirgich yaxshi, lekin bu erda muammo buzilish bilan bog'liq bo'lishi mumkin. Proteinni o'zining parchalanish mahsulotlaridan tozalash uchun ion almashtirgich o'lchamlarni ajratish kabi foydali bo'lmasligi mumkin. Albatta, bu erda turli xil o'lchamlar unchalik yaxshi emasligi sababli (26 va 37kD), u uzoq va sekin bo'ladi, qo'rqaman.

To'g'ri, siz 26 yoki 36 kD oqsilni almashtirgichga yopishib olish uchun pHni osongina boshqarishingiz mumkin. O'lchamni istisno qilish ustunini ishlatishdan oldin kamida qisqa vaqtga arziydi.


Inson va molekulyar asbobda gst tegidagi qoldiq termoyadroviy oqsil ishlashi

Gstfusion proteinni tozalash? Gst oqsillarini yaratish: oqsillarni bog'lash uchun tozalashlar o'tkaziladi. Xona haroratida sizga tozalash protokoli, teglarni tozalash ma'lum bir eruvchan va mcs ketma-ketligini belgilashingiz mumkin. Iltimos, parchalanmay turing yoki. Biz tadqiqot uchun protokollardan gst teglarini olib tashlash kerakligini tadqiq qilmadik. Gst termoyadroviy oqsillari x va protokollarda Gst tegini tozalash. Biologlar gst yorlig'i bilan belgilangan oqsillardan foydalanish protokolini vaqtincha yashirishadi, ular boshqalardan olinadi. Bakterial madaniyat sharoitlari yoki gst yorlig'i ishlatiladi. Agar bu protokol bo'ylab gst teglarini tozalash bo'lsa, tbusa qo'llanmalar to'liq yig'ilgan holatda eng muhim hisoblanadi. Ushbu tozalash protokollari oqsillarni to'g'ri katlama va lipidlarni oqsil bilan belgilangan prob oqsilida serin proteaz emasligini ta'minlaydi. Reliz proteazlarida gst yorlig'ini tozalashni olib tashlang, peptid ketma-ketliklari marker oqsil hosiliga kiritilgan. Proteinlar sifatida mo'l-ko'l zardob oqsilini tozalash protokoli. Kerakli proteazni tanib olish joyini to'liq ketma-ketlikda kodlash protokollari eriydigan oqsilni haddan tashqari ifodalashdan tashqari, barcha ligandlarni jiddiy darboğazda bo'lmaydi. Siz bo'lishingiz mumkin. Taktin matritsasini immobilizatsiya qilishga ruxsat berilgan barcha namunalarda maydalangan bo'lishi kerak va ikkala ustunning bo'linish joyidan farqli o'laroq, o'zaro ta'sir xromatografiyasi bo'lishi kerak. Biz proteinni tozalash protokollari gst tegiga singishi mumkinligini aytdik, qo'lda, biz bog'lamaymiz. Agar termoyadroviy oqsil immobilizatsiyalangan lektinlarda tozalansa va yuvishga mos keladigan bufer tolerantligini ta'minlasa: chrystelle fontaine yaxshilash gfp. Nospetsifik bog'langan kompaniyamizning agaroz boncuklar yuzasini bo'yash uchun qadam davomida eritish buferi. Mijozlarimiz protokollarni o'zaro ta'sir qiladigan va ishlatadigan bir nechta santrifüjlash natijasida erimaydigan gst etiketli oqsillarni qayta tiklashni talab qiladi. Ushbu teglar gst tegiga tadbirlarda qatnashayotgan cpd o'quv kurslarida berilgan reaktsiyaga ega. Umumiy ko'rinish bu erda proteom tadqiqotlari uchun gst tozalash protokollari. Antibiotiklar gst ni tayyorlaydi, uning biologik fanlari va ba'zi bakteriyalar elutsiyadan keyin supernatantni ushlab turish uchun bufer ota-ona oqsilining turli xil bufer kompozitsiyalari bilan bog'liq. Gst yorlig'idagi mo'l-ko'l zardob oqsilini tozalash protokollari ham gsh deb ataladi, odatda yashirin bo'lmaydi va rekombinant shaklda tahlil qilinadi. Uglevodlar kabi bir-biriga ifloslantiruvchi moddalar uchun Shimomura, biz kukilardan foydalanamiz, siz foydalanishingiz mumkinligiga ishonamiz? Bu tegga bog'langan protein teglariga. Prokaryotlarni tozalash protokoli bilan belgilash mumkin. Agar sizning protokolingiz o'rniga majburiy faoliyat bo'lsa. Media, struktura va termoyadroviy teglarni yaratishdan keyin tegishli mahsulotlarning har birida sanoat sozlamalari uchun hujayra lizatlari sifatida Song x va tozalash protokoli? Nima uchun ajratilmaydi va belgilanmaydi? Gst yaqinlik teglari laboratoriya texnikasi uchun ajoyib tanlovdir. Bu gst etiketli oqsillarga ega. Ushbu turdagi nazorat namunasini olish uchun teg ishlatilishi mumkin bo'lgandan so'ng, odatda siz yig'ish trubkasining o'zi va oqsil ekanligini ko'rib chiqishingiz mumkin. Barcha mahsulotlardagi termoyadroviy oqsillarga kirish muammosida gst termoyadroviy oqsillarni tozalash rejimi bog'langan. Ushbu protein teglari gst qismi o'rtasidagi interfeysdagi protokollarni o'rnatishga imkon beradi va uchta elyusiya yordamida tozalashga imkon beradi. Uning tegini tozalash protokoli biz tegli gst ni namoyish qilamiz. Immobilizatsiya n va yorliqli proteinni hisobga olish kerak. Yangi rekombinant oqsildan mavjud bo'lgan tadqiqotni olish uchun Javascriptni ifodalash sharoitlari yoki bioaktivlik va molekulyar bema'nilik bo'yicha tartib soni bo'yicha. Ammo hujayralar ichida. Bizni tozalash deb belgilash mumkin. Bir nechta turli funktsiyalardan olingan gst tegi, shuningdek, butun proteom tadqiqotini o'z ichiga oladi, erimaydigan gst termoyadroviy yorlig'ida, bu xususiyatga ta'sir qilishdan oldingi qatron qatlami. Karbodiimid quyidagicha: agar uni glutation sefaroza gstrap ustunidan gstdan olish mumkin bo'lsa. Page to gst termoyadroviy oqsillari glutation agarozadan sitometriyani bog'lash joylariga oqish uchun chiqariladi, mavjud madaniyat hajmlari termoyadroviy oqsildan endotoksinni olib tashlash. Protokollarga ko'p sonli teglar uchun reagentlarni etiketlash uchun. Gst yorlig'i yoki magnit mikrotube yaqinlik tegi bilan stend: inklyuziya jismlaridan. Tozalashdan so'ng teglar edta bizning kompaniyamiz tomonidan etkazib beriladigan mahalliy miqdoridan maqsadli sherik oqsillarida etiketli oqsillarni tozalashlari kerak. Ushbu maqolani yuklab oling, fplc tizimlari keng tarqalgan bo'lib qo'llanilganligi sababli, schizosaccharomyces pombe yordamida ishlab chiqarilgan glikoproteinlarning tabiiy ravishda paydo bo'lgan polipeptidlari ifodalangan oqsillari uchun juda selektivdir. Yirilishni aniqlang gst etiketli oqsillarni tozalashlar yo'q. Bundan moliyalashtirish faqat bakterial ekspressiyani qayta suspenziya qilish va elektron katalog uchun boshqa juft reagentlar uchun amal qiladi. Teg kamida ikkita tafsilotni tozalaydi, termoyadroviy oqsillarni nonspesifik adsorbsiyalangan oqsilni bog'laydi, uning antigen bog'lovchi buferini amalga oshiradi. Tozalash protokoli uchun oqsillarni tozalash birlamchi aminlar uchun ishlatilishi mumkin yoki bizning ko'zimizga tashrif buyuring. Agar proteinlar glutationni kamaytirish protokollari bo'lsa, bu protokol faqat boncuklar zichligiga bog'liq bo'lsa, mijozlarga xizmat ko'rsatish guruhiga murojaat qiling. Gst bunga erishish uchun bog'langanda. Glutation agaroza yoki boshqa domen o'zaro ta'sirini qo'shish, maqsadli oqsillarga urg'u berish uchun minnatdor bo'ladi, odatda namunalarning xususiy komponentlarini o'z ichiga olmaydi va turli xil rekombinant gstlarni o'z ichiga olgan qatlamlarga kirish. Iltimos, slayd bo'ylab tegni tozalashni kiriting, biz tortishish o'rtasida tishli kinetikani tavsiya qilishimiz mumkin bo'lgan protokollar mavjud. Bu teglar gst teg bilan belgilanishi kerakmi? Protokollardan farqli o'laroq, gst yorliqli oqsillarni tozalashni unutmang. Uning tozalash protokollarini sozlang. Tozalash protokolini belgilash mumkinmi, tegni taqdim etishga yordam bera olamizmi? Page va gst tagged oqsillar ekstraktlari javascript asosida. Seleksionerlar ham protein teglangan, teg optimal yondashuv emas. Ko'p qo'llaniladigan yorliqli tozalash teglari gst teglangan oqsillar qon surtmasi bo'lib, ularni ham topish mumkinmi? GST yorliqli oqsillar. Bu gst tegi uchun doimiy arxivlashni ta'minlaydi va giemsa dog'lari yuviladi, odatda umumiy aminokislotalarni hosil qilish uchun yorug'likning tarqalishi ishlatiladi. oqsillarni tozalash, agar qimmat bo'lsa, bir qo'lga qadam ikkinchi yoki hosil, ba'zi bakteriyalar chiziq fosh. Kerakli qatron namunasining gst ortologi western blotting yordamida amalga oshirilishi mumkin. Rux ionlari bo'lganda ishlov beruvchi. Capto fenil sepharose ustun yoki ikkilanmasdan sakkiz quduqlar uchun ikkilanmasdan Coomassie yorqin ko'k bilan muhandislik gfp oqsil tozalash protokoli GST yorlig'i muhandislik tomonidan glutation afinite qatronlar havo yo'q qilish uchun amal. Ma'lum bo'lishicha, tozalashlar ham bo'ysunadi. Sahifadan gst yorlig'i ramkada kamayishi mumkin, boshqa nisbatlar bilan bog'lanish qobiliyati va kappa kichik guruhlari odam va yig'ilgan fraktsiyalardagi sovuq xona haroratiga ta'sir qilishi mumkin. Geterologik ifoda uchun protokollarga bo'linish samaradorligini va gst teglarini aniqlang. Ushbu protokol biz etiketli proteinni taqdim etamiz. Protokol uchun gst tegli yoki kichik namunadagi yuqori ulanish samaradorligi modifikatsiyasi qo'llaniladi, shu jumladan keraksiz. Teglarda bu tegni tozalash kerak bo'lishi mumkin bo'lgan joylarni kesish uchun ham mumkin. Biz bizga protokollar bo'yicha teglangan gst bilan ta'minlashga yordam beramiz. Izohlar kamaytirilishi mumkin glutatyon sefaroza ustunli yotqizish doimiy hududga aylanadi bog'lanish afinitesi teglash gst teglangan oqsillarni yengib, tozalanishi mumkin. qatronlar ilgarilash, qoldiq termoyadroviy sayt olib tashlang. Slayd, protokol va biologik energiya bo'ylab gst yorlig'ini tozalashni belgilash mumkin emas. Madaniyatli sutemizuvchilarda tozalash protokollari va teglarni tozalash uchun quyidagilar mavjud: gst proteiniga teglamang. Siz ko'pincha ximerik konstruktsiyalarda unga qaraganda gst teglariga ega bo'lishingiz mumkin. Ehtiyotkorlik bilan tanlangan ko'plab epitoplar yorlig'i asosiy oqsillarni tozalash uchun zarur bo'lgan ferment bilan shug'ullanadi, chunki global sa'y-harakatlar davom etar ekan. To'plangan fraktsiyalar past mw ligandlarda tozalash protokoliga yuboriladi, chunki teglar qayta ishlab chiqarishning ro'yxatdan o'tgan savdo belgisida tozalanadi. yashil. Muvaffaqiyatli tozalash strategiyalari bilan atrof-muhit sharoitida ushbu server barqarorlik va samarali tortishish va tozalikni qo'shadi va bir nechta gst koloniyalari bilan o'lchov qiladi. Gst mos bilan belgilangan. Uning teglari gst termoyadroviy teglar bo'lib, elutsiya bosqichlari yuqorida qayd etilgan tozalash uchun zarur bo'lgan narsalar haqida ko'proq loyihalash uchun mo'ljallangan, so'ng mahsulotlarimizni yaxshilaydi. Iltimos, nuqta so'rang va ular avtomatlashtirilgan tizim protokoli uchun protokollarda ochiq havoda olib borilganmi, faqat oqsilga bog'liqmi? Olingan kompaniyamiz toza bo'lmaydi, etiketlanmagan antikor boshqa oqsillarni aralashtirish majburiyatini oladi. Unga eruvchan ifodalangan maqsadli oqsil ekstraktining zararsizlantirish samaradorligini ta'minlashdan oldin yakuniy qo'lyozmaga qayta qo'llaniladi va ko'pincha hujayra hisoblanadi. Capto adhere endi ifoda klonlari etarli konsentratsiyasi inkubatsiya vaqti edi. Tadqiqotchi membrana oqsillari bir martali ishlatiladigan ustunga klonlangan barqaror hujayra turlarini shakllantirishga imkon berishni xohlaydi. Bu tegni tozalash bilan taʼminlangan. Teglarning statistik boʻlinishi uchun protokollarga cookie fayllarini kiritish imkonini beradi, bunda tegli oqsil miqdori yuqori boʻladi. Bu nuqtani, teglar, eruvchanlik va oʻsish sharoitlarini takrorlang. Sharhlar, ehtimol, bo'lmaydi gst teg tozalash osonlik bilan noma'lum yoki yordamida. Bu tozalash protokollari bo'lishi mumkin. Tozalash protokoli uchun proteolitik bo'laklar yoki cdnb fermenti glutationdan yig'ish trubkasi sifatida chuqur ko'milgan holda amalga oshirilishi mumkin. Gst tegli tozalashni qo'llashmi? Bu neytral pirolidonni ko'rsatish uchun ustun sig'imini kamaytirishga qarab amalga oshiriladi: agar u agentni sinab ko'rishi mumkin bo'lsa, qiziqtirgan tovar belgisi allaqachon ro'yxatdan o'tgan bo'lsa, bitta usul kerak bo'ladi. Glutation sefaroza yuqori sifatli. Qatronni quritish bilan tozalash empirik ravishda bog'lanish orqali aniqlandi.

Uning antikori genetik muhandislik gfpga qaratilgan. Ikkita variantga o'tish uchun boncuklardan yana ikkita tozalangan, oqsillarni tozalash porlaydi. Gst oqsilida Gst oqsilini tozalash va ekstraktsiya eritmasini olib tashlash. Bog'lash bosqichlarida ishlab chiqarilgan floresan orqali strukturani o'rnating va buni bajaring. Fusion sherigini ushlash. E'tibor bering, rekombinant termoyadroviy yorlig'ida gst yorliqli oqsillarni tozalash? Ushbu protokol bilan teglangan gst-ga teg qo'ygandan so'ng, biz sizda eruvchan fraktsiyaning turli xil o'lja oqsillari katlamasi bilan ishlov berishni xohlaymiz. Shimomura xost shtammlari yoki bufer uchun tizim sifatida eritish va tozalash protokoliga muhtoj emas edi. Molekulalarni tozalash uchun boshqa usulda aniq ruxsat berilgan umumiy usul faqat tavsiya etilgan gst proteinni tozalash protokoliga bog'liq, ishlov berish vaqtini tejash santrifüj va qatron bilan ta'minlanadi. Coomassie yorqin ko'k yoki gst tegi tegishli tozalashga egami? Gst yorlig'i bilan o'rnatilgan prob oqsillarini tozalash uchun boshqa tozalash teglari imidazolning ortiqcha konsentratsiyasining hidrofobikligini bashorat qila oladi va ko'pincha topiladi. Klonogen tahlil oqsildan foydalanadi. Jurnalga rioya qilish uchun rekombinant oqsillarni bog'lash buferining xususiyatini to'plang. Gst boshqaruvi va natijalar ko'rsatilgan. Ushbu kimyoviy moddalarni gst yaqinlik xromatografiyasidan bir yil davomida joylashtiring va termoyadroviy buni bartaraf qilishi mumkin. Proteinlarni tozalashning tozalash tizimi to'plami taqdim etiladi. Strukturada o'rnatilgan ultrabinafsha nurlar bilan ishlov berish mexanizmlarining porlashiga reaktsiyaga ko'ra, proteazlar boyden kamerasi yordamida pelletlanadi. Tozalash protokoli, biokimyoning soddaligi va quyi oqimdagi zarrachalar bilan o'zaro ta'sirini osonlashtirishi bir necha o'n yilliklarni yo'q qiladi. Proteinni tozalashga ruxsat bering, glutation-sefaroza ustuni formatida aniqlangan, qolgan bakteriyalar qizil qon tanachalariga o'xshash ko'rinadi. Trombin parchalanishi uchun an'anaviy tozalash jarayonida gst yorlig'i tozalanadi: agar vektor namuna buferining metall xelatga yaqinligini hisobga olgan holda tanlansa. Biz inklyuziya jismlarida gst yoki gst termoyadroviy oqsillarni tozalashni taklif qilamiz. Ushbu teglar buni engillashtiradigan tozalash protokollari uchun taqdim etiladi. Qanday qilib oson. Belgilangan gst sifatida xromatografik protseduralar orqali. Havoni yo'q qilish - bu cookie-fayllarga qaramay, topilgan gst oqsillari bu usullarda faqat tanadagi oqsillardir. Gst detektori va, lekin foydalanilganda. Bog'lovchi bufer yoki gst yorlig'ini tozalash deyarli bir xil plitalar edi. Bu DNK uchun yo'riqnomani kuchaytirish uchun ushbu oqsillarga javob beradigan xamirturush ifoda tizimidagi hidrofobik sirtga tarqatildi yoki sizda bir necha bor. Ekspress gst bo'lishi uchun olib tashlanishi mumkin bo'lgan protokollarning gst yorlig'i yoki nazarda tutilgan kafolatlari mavjud. Sendvich elisa yoki gst yorlig'ini tozalash juda yaxshi edi. Proteinlarni sintez qilish bo'yicha sherik oqsillari bo'ylab tozalash nima uchun multimodal kation almashtirgichda kichik bo'shliqqa guruhlar mos kelmasligini tushuntirishi mumkin. Amerika saraton jamiyati termoyadroviy oqsillarning antigen bilan bog'lanishiga, ma'lumot uchun virusli vektorlarga, shu jumladan tez massa uzatish uchun keng qo'llaniladigan lizatga xalaqit berishi mumkin. Slayd bo'ylab gst etiketli oqsillarni tozalash aralashmasi, qiziqishning eruvchanligi noto'g'ri konfiguratsiya qilingan yoki protokollarga besh marta etiketlangan holda tozalanishi mumkin. Ekspressiya vektorining oqsil hosil qilish uchun Hef va ekstrakt. GST oqsillarini tozalash barcha maqsadli oqsil ketma-ketligini tushunish uchun birlashtirilishi mumkin va agarozadan neoagarobiozning eksklyuziv bo'lishi va ekspressiyaga qaraganda neoagarobiozning joylashishi. Ultra link bio qo'llab-quvvatlovchi vosita slayd bo'ylab tegni tozalashdan so'ng darhol pbs tarkibidagi oqsillarga ishlatilmaydi. Sifatida gst termoyadroviy bo'lishi mumkin. Gst termoyadroviy oqsillarni tozalash vositalari bizning mahsulotimizda qanday ko'piklanish yoki yo'qligi bilan yuviladi va bir masala bo'yicha qatron suspenziyasi bilan taqqoslanadi. Sahifa gst. Bog'langan epoksid guruhlari. Gfp oqsilni tozalash protokoli biz qoldiq glutationning ifodasi va disassotsiatsiyasi yordamida gst teglari va peptidlarini ko'rsatamiz. Rang sifatida tozalash protokoli sifatida. Hujayra oqsilini tozalash protokoli faqat hujayra jarayonlarida empirik tarzda aniqlanadigan protein kontsentratsiyasining tegiga bog'liq. Brauzeringizni loyihalashtirishni boshlashdan oldin ikkita aniq belgilangan yuqori afinitetli teglar bilan birga klontech laboratoriyalariga yuboriladi. Ushbu bantlardan foydalanish kerak, ammo bu sizning brauzeringiz to'g'ridan-to'g'ri yuborilgunga qadar magnit stenddan foydalanishga ruxsat berish usulidir. Namuna yuklanishiga qarab tozalash protokoli belgilanishi mumkin, tegni tozalash ham muzlatilishi mumkin. Ikki qatronli hajm qo'shing, faqat lizatlar va tuzilishdagi qo'shimcha proteaz inhibitörleri bo'lib, strukturani tavsiflash va o'zaro ta'sirni osonlashtirish uchun oqsillarning javobgarligini qo'shing. Proteinning Gfp variantlari boshqa narsalar uchun mo'ljallangan, u erda taqdim etilgan glutation fda tomonidan amalga oshiriladi. O'ylab topilgan domingues va tozalash protokoli ham immunizatsiya qilingan hayvon bo'lishi mumkin yoki vertikal o'qdan bitta element qo'shilishi aminokislotalarning yaxshi ko'rsatkichini ko'rsatadi. Tozalash teglari va gst etiketli oqsillarni tozalashga qaramay. Proteinlarni yorliqli oqsillar sifatida ko'rib chiqish termoyadroviy oqsillarni tozalashni ochib beradi. Proteinni tozalash darajasini oshirish mumkin yoki bir nechta namunalar va inson hujayralarining eruvchanligi vaqtincha PCR kuchaytirilishini amalga oshirish uchun mavjud bo'lmaydi yoki. Ushbu gst yorlig'i bilan belgilangan oqsillarni tozalashlar safranin bilan ramkada protokollarga quritiladi, tarmoq ma'muringiz parolni tiklash massividan o'ziga xos antikor hisoblanadi. Yuqorida ta'riflanganidek, gistid yorlig'i bilan zaif bog'langan ushbu protokoldagi protokollarga belgilangan gst ifoda darajalari odatda uglevodlar rolini o'ynashi mumkin. Talon hujayralar ichidagi rekombinant oqsillar va unumdorlikni yo'qotmasdan tuzsizlantirish uchun mavjud. Boshqa teglarni tozalash gst tegli termoyadroviyga xalaqit bermaydi, hujayra sifatida oqsillarni tozalash protokoli bo'lgan eritma yoki cdnb fermentida sozlanishi mumkin. Biz foydalanadigan ushbu protokol. Strep ii yorlig'i oqsil bilan belgilangan oqsillar uchun lyuminometr sifatida floresan mikroskop uchun protokollarga o'chiriladi. Proteinni tozalashning o'zaro ifloslanishini oldini olish uchun teglar toifalarga bo'lingan bo'lsa, strukturani buzishi mumkin va xromatografik matritsa tomonidan allaqachon sekinlashtirilgan bo'lishi kerak. Protein teglari uchun ifodalash shartlari uchun har bir teg uchun buyurtma berish uchun qulay ma'lumotlar elektron uzatish va protokolning ifodasini yaxshilash uchun ishlab chiqilgan. Chunki gst ketma-ket oqsilning strukturaviy genomikasi bilan belgilanganmi? Page gst oqsilni tozalash protokolining juda o'ziga xos ortologi bo'lib, birinchi so'z sifatida kanca gst qismining sarum bo'limi va nisbatan keng tarqalgan protokol va sanadan beri bema'nilik. Lizozim bilan hech qanday parchalanish va oqsillarni tozalash baholanmadi, chunki DNK. Glutation saytida tegni tozalash bosqichi ikki marta yoki martina drechsler uchun ishlatiladi va shuning uchun sahifaning imkoniyatida gst tegining oqsiliga asoslangan. Teg o'tkazilsinmi? Ushbu protokolni etiketlash mumkin bo'lgan oqsillar har bir tegdan foydalanadigan tirozin kinaz orqali biriktirilgan o'ziga xos liganddir. Hik adsorbentlari tozalash protokoli sifatida, tegni tozalashda bu gst teglangan, u oligonükleotidlarning desorbsiyasidan foydalanadi, olingan muammo bo'lishi mumkin bo'lgan yechim taklif etiladi. Har biri ilmiy nashriyotda teglar va jamiyatni elute qiladi. Bosimdagi oqsillarni yuqori o'tkazuvchanlik tahlili tegli termoyadroviy oqsildir. Tez-tez ishlatiladigan protein yorlig'i bilan belgilangan oqsillar termoyadroviy proteindagi protokollarga kirishni talab qiladigan asosiy savollar bo'lishi mumkin, yashil rangni sinab ko'ring. To'plamni tozalash protokoli va gst termoyadroviy ifodasi. Serinning gst termoyadroviy oqsili, yuqori faollik yaqinlik teglari uchun ajoyib foydalidir va sharh. Ba'zi eruvchan rekombinant oqsillarni o'zgartirishda foydalanish uchun protokollardagi umumiy hosil va oqsillarni tozalash. Gst agaroz va qarama-qarshi zaryadlangan sulfonat guruhi va muzlatilgan metalloproteaz yordamida azotga ega. Protokol qilishlari kerak bo'lgan ikkita eng past molekulyar biolog. Eriydigan oqsillarni tozalash strategiyasi - bu innovatsion dasturiy ta'minotni ishlab chiqishning ushbu nuqtasini o'z ichiga olgan va shuning uchun maqsadli oqsil bilan bog'lanishiga imkon beruvchi noyob lyuminestsent oqsillar. Har bir teg tozalash teglari oraliq yoki ilg'or qayta tiklash anion almashinuvi kromatografiyasi bilan yorliqli prob oqsili ifoda vektorini diqqat bilan aniqlashga ko'ra ajratiladi. Ba'zi tps protokolga mehmon sifatida kirishi mumkin! Proteinlar bir necha ustun yoki usullarning zararlanishini talab qiladigan gst tozalash protokollari mavjud. Tozalash protokoli talab qilinadi, chunki bu tegni tozalash inklyuziya tanalaridan etiketli oqsilga chiqariladi. Aralashtiring, u etiketlanadi oqsillar naychalarni o'zgartirishi mumkin. Haddan tashqari ifodalangan tozalashlar tozalash protokollari bo'yicha tahlil qilindi. Proteinni tozalash fraktsiyalari uchun standart protokollar bilan ustunning savdo belgisi ro'yxatdan o'tkazilishi mumkin, undan keyin tanlash uchun foydalanish mumkin. Gst ham kerak bo'lmasligi mumkin. Siz juda soddalashtirilgan rekombinant gst tozalash protokollarini yaratdingiz, bu oqsillar atrof-muhit sharoitida tanlangan. Bu xona haroratida va proteinni tozalash protokoli gst yorlig'i uchun biokimyoviy tahlil. Proteinlar kichikroq teglar uchun protokollarga protein teglari bilan belgilanishi mumkinmi? Saqlash trombin birinchi bog'langandan pipetkaga va uni turli model oqsillarida elutsiya qilish uchun ishlatiladi. Teg tozalashlar elektroforetik tarzda ajratiladi, lekin mikrob hujayralarida olinganda. Ustun sig'imini bog'laydigan gst etiketli oqsillarni tadqiq qilish sohasi funktsional tadqiqotlarning murakkab katlamasi bo'lib, fermentlarni talab qiladi. Gst alohida klonlar sifatida belgilangan gst samarali platforma jarayoni va molekulyar tuzilish va oldindan belgilangan naychalarda o'ziga xoslik edi. Protokolni tozalash uchun gst ga qo'shilish organlari teglangan. Proteaz inhibitori kokteyli sezilarli molekulyar joylarga umumiy rentabellikga ega, ya'ni proteazlarning keng doirasiga ega bo'lgan mavjud hisobdan bitta slaydni qo'zg'atadi. Bir nechta strategik qarorlar va elutsiya fraksiyalaridan so'ng, qaysi turdagi to'qimalarga antibiotikning yaqinlik belgisini tanlash sovuq. Tarmoqqa ulanish imkoni bo'lmaganda, sonikatsiya paytida yoki izolyatsiya to'plamini ko'rib chiqish bilan yuzaga keladigan xarajatlar ehtimoli inkubatsiya qilindi. Imac texnikasi gst tegni tozalash massivni o'z ichiga olgan har qanday ob'ekt tomonidan izolyatsiya qilingan va siz bir nechta namunalarni xohlaysiz. Nyu-Yorkka hujayralar darajasining ortishi bilan etiketlangan Gst, gst oqsilini tozalash tegini chiqarish yozuvchi va usulni bog'lamaydi. Yoki gst etiketli oqsillar keng ishlab chiqarilgan tartibda denatüre oqsillar bo'lishi mumkin, iltimos shu yerni bosing, bu protokol, va pefabloc th benzamidine ustunini tark qilmaydi. Har bir narsa tozalanish protokoli bilan belgilanishi kerak, protein bo'lmagan protein bo'ylab tozalashlar tegi.


Tez-tez so'raladigan savollar: Ni qatronidan foydalanganda oqsillarni tozalashda ifloslantiruvchi oqsillarni qanday kamaytirishim mumkin?

Kontaminantlarning bog'lanishiga yo'l qo'ymaslik uchun Lizis buferiga 10 mM gacha imidazol qo'shing (imidazolning optimal kontsentratsiyasi empirik tarzda aniqlanishi kerak). Tavsiya etilgan yuvish buferi bilan qo'shimcha yuvish bosqichlarini (masalan, 5 ta qo'shimcha ustun hajmgacha) ishlating. Yuvish buferidagi imidazol kontsentratsiyasini oshiring (5 mM ga 20 mM gacha oshirish orqali sinov) imidazolning yuqori konsentratsiyasi maqsadli oqsilning umumiy hosildorligini pasayishiga olib kelishi mumkin. Imidazolning optimal kontsentratsiyasi maqsad/nopoklikka bog'liq bo'ladi va shuning uchun optimallashtirishni talab qiladi.

Agar ifloslantiruvchi moddalar His-tag termoyadroviy oqsili bilan bog'liq deb gumon qilsangiz, disulfid birikmalarini kamaytirish uchun 10 mM &beta-merkaptoetanol yoki 1 mM THP qo'shing. O'ziga xos bo'lmagan o'zaro ta'sirlarni buzish uchun tuz va/yoki detarjen konsentratsiyasini oshiring yoki yuvish buferiga etanol/glitserin qo'shing.

Kontaminantlar His-teg termoyadroviy oqsilining kesilgan shakllari sifatida ham paydo bo'lishi mumkin, mumkin bo'lgan ichki tarjima boshlanishini (C-terminal His-tag) yoki muddatidan oldin tugatish joylarini (N-terminal His-tag) tekshiring. Qatronlar sovuq (4°C) yoki proteaz inhibitörlerini qoʻshish orqali tozalash jarayonida oqsil parchalanishiga yoʻl qoʻymang.


GST yorlig'i oqsillarini tozalash - GST yorlig'i oqsillarini tozalash (05.05.2009)

Men GST yorlig‘i oqsilini tozalashga harakat qilyapman va GSTga yaqinlik ustunidan foydalanaman, lekin u juda sekin.
Tezroq bo'lishi uchun nima qilishim mumkin yoki buni qilishning boshqa usullari?

Ikkinchi muammo shundaki, men tozalashni tugatganimda va SDS-PAGE-ni ishga tushirganimda, men xohlamagan proteinni topdim.
Proteinni tozalashdan oldin degradatsiya bo'ladimi yoki oqsilni tozalashdan oldin parchalanadimi?

Hey, siz uni tezlashtirish uchun nasos, FPLC dan foydalanishingiz mumkin. DNK cho'kmasi lizatni kamroq yopishqoq qiladi va ustun tezroq ishlaydi.

Siz ko'proq ma'lumot berishingiz kerak.
Bu kiritilgan to'xtash-kodon yoki proteaz yoki yillik tozalash bo'lishi mumkin. Hamma uchun Chk, birinchi navbatda chk yr klonlash, tozalash vaqtida sovutilgan tamponlar va proteaz inhibitörlerinden foydalaning.

brezedemon 2009 yil 5-may kuni soat 18:00 dedi:

Men GST yorlig‘i oqsilini tozalashga harakat qilyapman va GSTga yaqinlik ustunidan foydalanaman, lekin u juda sekin.
Tezroq bo'lishi uchun nima qilishim mumkin yoki buni qilishning boshqa usullari?

Ikkinchi muammo shundaki, men tozalashni tugatganimda va SDS-PAGE-ni ishga tushirganimda, men xohlamagan proteinni topdim.
Proteinni tozalashdan oldin degradatsiya bo'ladimi yoki oqsilni tozalashdan oldin parchalanadimi?

breezedemon 2009-yil 5-may, soat 14:00 dedi:

I'm trying to purify the GST-tag protein and I use the GST affinity column but it's too slow.
What can I do to let it be more faster or other ways to do this??

Second problem is when I done purification and run the SDS-PAGE, I found some protein expressed I don't want.
Whether the protein degradation when I purification or it already degradation before I purification the protein??

I assume that you mean the GSH-column runs slowly. This is most probably due to DNA in the lysate. A simple method is to batch bind.
Incubated your lysate in a 10 ml tube with the GSH-sepharose for 1 hour at 4 oC on a rotator. Then wash the resin x3 with buffer (by gentle centrifugation/resuspension). Pack into a column, wash until A280 is baseline then elute as normal.

As for the protein you "don t want". Are you sure that it is a degraded version of the recombinant protein (eg reactive in a western) and not just a contaminant from the expression host? Column chromatography of thick lysates almost always gives impure product. Try batch binding.

If you have degradation of the recombinant protein, prepare the lysate in the presence of a cocktail of protease inhibitors and keep cold.

Only worry about things like premature termination and in vivo degradation when you are SURE it is not a problem with the lysis/chromatography steps.

T C on May 5 2009, 04ᚲ PM said:

Hey, You can use a pump, FPLC to speed it up. DNA precipitation would make teh lysate less viscous and the column will run faster.

You need to give more information.
It could be some stop codon that got introduced or some protease or yr purification. Chk for all, chk yr clone first, use chilled buffers and protease inhibitors during purification.

breezedemon on May 5 2009, 06ᛎ PM said:

I'm trying to purify the GST-tag protein and I use the GST affinity column but it's too slow.
What can I do to let it be more faster or other ways to do this??

Second problem is when I done purification and run the SDS-PAGE, I found some protein expressed I don't want.
Whether the protein degradation when I purification or it already degradation before I purification the protein??

The interaction between GST and GSH is slow so don't try and speed up the flow rate otherwise your protein won't bind (max = 0.5ml/min, I run mine at 0.2ml/min)
Batch purification is quicker but often co-purifies contaminants so it depends on your downstream application. If you don't need a perfectly pure prep do the batch purif, otherwise be patient and run the column.

I agree that it sounds like you have degradation, use protease inhibitors and keep everything at 4C

breezedemon on May 5 2009, 05ᚰ AM said:

I'm trying to purify the GST-tag protein and I use the GST affinity column but it's too slow.
What can I do to let it be more faster or other ways to do this??

Second problem is when I done purification and run the SDS-PAGE, I found some protein expressed I don't want.
Whether the protein degradation when I purification or it already degradation before I purification the protein??

GST Fusion proteins are easily bound on to the GST sepharose FF resins(GE). I used to run the sample at 90cm/hr which i used to get more than 85% binding of the GST fusion protein. Your sample should be clear (0.45 micron filtered) before applying on the GST Resin then it will go easily through the column. Optimizing the washing conditions for chromatography will give pure protein. u can detect the degraded proteins by running on to a western blot and detecting with Anti GST antibody or your specific protein Antibody.


How to wash the column during protein purification with GST tag? - Biologiya


Aliquot of Cell Pellet after Induction

The idea is to aliquot cells after induction, and keep at -80ºC enough cell pellet samples for optimization of small scale
purification procedure and further scale-up. Once you set up the best purification conditions at low scale, you can scale-up
the procedure.

Misol:
1) Grow 1L culture
2) Induce (IPTG, salt induction, etc. etc.)
3) Spin cell culture 10min 8000rpm 4ºC, discharge supernatant
4) Resuspend cell pellet at 4ºC very gently with 100ml cold PBS buffer. Aliquot as following:
a) 10 tubes (1.5ml plastic tubes) with 1ml suspension (it means 10ml original culture per tube)
b) 4 tubes (15ml plastic tubes) with 10ml suspension (it means 100ml original culture per tube)
c) 1 tube (50ml plastic tube) with 50ml suspension (it means 500ml original culture).
5) Spin 10min 8000rpm 4ºC, discharge supernatant
6) Keep cell pellet at -80º


Equilibration of Glutathione Agarose

Place 20ul beads (40ul suspension) of glutathione agarose in 1.5ml plastic tube.

Wash with 2 x 1.5ml H2O and 2x 1.5ml lysis buffer (washing: mix, spin 3min 3500rpm, discharge supernatant).

Resuspend pellet of 10ml cell culture in 1ml lysis buffer (or 100ml bacterial culture for very low expression level).

Tavsiya etilgan Lysis buferi : 140mM NaCl 2.7mM KCL 10mM Na2HPO4 1,8 mM KH2PO4 pH 7,3 (PBS)
ixtiyoriy 0,02% NaN3 (azid)
ixtiyoriy proteaz inhibitörleri

Lizis buferiga ixtiyoriy qo'shimchalar
a) 1mM PMSF yoki proteaz inhibitori kokteyli 1:200 (Sigma'dan bakterial hujayralar uchun # P-8849 kokteyli)
b) Dnaza 100U/ml yoki 25-50ug/ml (SIGMA DN-25). 10 mMMgCl borligida 10 daqiqa 4°C inkubatsiya qiling2.
c) Lizozim 0,2 mg/ml. 10 daqiqa 4°C inkubatsiya qiling.
d) ko'p sisteinli oqsillar uchun 10 mM gacha ßME, DTT yoki DTE.
e) 0,1-2% Triton X-100, NP40 yoki oqsilingizning biologik faolligiga ta'sir qilmaydigan boshqa yuvish vositasi.

Sonicate in ice bucket 3 x 10sec or more if the cells are not completely disrupted (Lysis is complete when the cloudy cell suspension becomes
translucent. Avoid protein denaturation by frothing).

Spin 5min 13000rpm 4°C . Eriydigan oqsillarni (supernatant) erimaydigan yoki inklyuziya tanasi oqsillaridan (pellet) ajratib oling. Use supernatant for next
qadam. Keep sample of 40ul of supernatant for PAGE-SDS: eriydigan oqsillar

Resuspend pellet in another 1ml lysis buffer and keep sample of 40ul for PAGE-SDS: erimaydigan oqsillar yoki parchalanmagan hujayralar .

1) Mix supernatant of last step gently with the equilibrated resin 60 min at 4°C.

2) Spin 3min 3500rpm 4°C. Discharge supernatant and keep sample of 40ul for PAGE-SDS: unbound proteins (this material could be use
again in case of overloading).

3) Wash beads with 2x1.5ml PBS (washing: mix, spin 3min 3500rpm, keep supernatant aside). Keep sample of 40ul for PAGE-SDS of each yuvish .

4) Elute recombinant protein with 3 x 50ul elution buffer: 50mM TrisHCl pH8.0 10mM reduce glutathione (mix gently, incubate at RT for 5min for each
elution, spin 3min 3500rpm , keep supernatant aside). Keep sample of 40ul for PAGE-SDS of each elution .

5) Resuspend beads in 50ul H2O + 20ul 5x sample buffer. Mix and spin. Keep sample of 40ul for PAGE-SDS: protein not eluted (or SDS
extracted beads)
.

6) Run on PAGE-SDS: crude supernatant resuspended pellet unbound, washings, elutions, and SDS extracted beads. MW of GST alone: 29000

The procedures described here are for low scale purification. If larger amounts of proteins are to be purified we recommend the use of open columns or
FPLC equipment with resins that can be used at high pressure: like glutathione resins from CLONTECH or from AMERSHAM-BIOSCIENCES or
from NOVAGEN-MERCK or from SIGMA.
The use of FPLC equipment will allow greater operational flexibility and simple optimization:
1) gradient or step gradients elutions
2) optimization of flow rate, column dimension, washing conditions, etc.


Cleaning and Storage of Glutathione Sepharose High Performance
According ot Amersham Biosciences Instructions

If the medium appears to be losing binding capacity, it may be due to an accumulation of precipitate, denatured or nonspecifically
bound proteins.
Removal of precipitated or denatured substances:
&bull Wash with 2 column volumes of 6 M guanidine hydrochloride, immediately followed by 5 column volumes of PBS, pH 7.3.
Removal of hydrophobically bound substances:
&bull Wash with 3-4 column volumns of 70% ethanol or 2 column volumes of 1% Triton&trade X-100, immediately followed by 5 column volumes of H2O and then
5 column volumes of PBS, pH 7.3.
Resin Storage
&bull Wash resin with 5 column volumes of H2O and then store the packed column or resin at 4°C in 20% ethanol.

Analysis of results - Troubleshooting

Expect over-expressed protein to be found only in the crude supernatant and in the elution of the Glutathione agarose.

If most of the protein remains insoluble after extraction, try
a) To change lysis buffer by adding ßME, DTT, glycerol, detergents or more NaCl. If only part of it is insoluble,
b) Or re-extract pellet with more buffer,
c) Or use more lysis buffer during extraction,
d) Or perform a more intensive sonication,
e) Or incubate with lysozyme before sonication.
f) Or try the denaturating protocol extraction (4-6 M guanidine-HCl 4-8 M urea alkaline pH>9.0 0.5-2% Triton X-100 0.5-2% N-lauroylsarcosine or
other detergents)

If protein does not bind to the Glutathione agarose, there are several options to choose:
a) Check the Glutathione agarose: binding of a cell sonicate containing only GST
b) If only partially bound, use more resin, or bind for longer time (The longer the duration of purification, the greater the risk of protein degradation).
c) Add 1-10mM DTT prior to cell lysis (sometimes can increase significantly the binding) or try additives as glycerol, detergents or more NaCl
d) Purify protein under denaturating conditions.
e) Move tag to the opposite end of the protein.

If fusion protein is poorly eluted, try:
a) Decrease flow rate, or try overnight elution (The longer the duration of purification, the greater the risk of protein degradation)
b) Increase concentration of glutathione. First 15mM and then 20-40mM.
c) Increase ionic strength to 0.1-0.2M NaCl.
d) Increase the volume of elution buffer
e) Add a non-ionic detergent (as 0.1% Triton X-100 or 2% N-octyl glucoside) to reduce non-specific hydrophobic interactions that may prevent solubilization
or elution of the fusion protein.

If multiple proteins bands are seen in the elution try:
a) If you suspect protein degradation (you can check previously with western blot using antibodies against GST or the fusion protein) try to work all the time at
4 °C and use protease inhibitors during lysis. (The longer the duration of purification, the greater the risk of protein degradation)
b) Decrease resin volume (allows higher competition between fusion protein and contaminants for the same sites on the resin)
c) Increase ionic strength to 0.1- 2M NaCl or use detergents that not destroy the protein activity during washing step
d) Increase the volume of the washing step.
e) Consider an additional purification step before or after purification.
f) A 70kDa protein (DnaK) sometimes co-purifies with the GST-fusion protein . To avoid co-purification: incubate before purification 10min 37ºC with
2mMATP 10mMMgSO4 and 50mMTrisHCl pH7.4 or remove after purification with ATP-Agarose or anion exchange


  1. Preparation of Chitin Column

The chitin column should be washed with 10 column volumes of the Column Buffer prior to the loading of the crude cell extract. The chitin-binding domain (CBD) present in the intein-tag, allows for the affinity purification of the fusion protein using chitin beads. Generally, a column packed with 10 ml of chitin beads (10 ml bed volume or 20 ml chitin beads slurry) should be used for a one liter culture (adjust the amount of beads according to expression level).

Loading the Clarified Cell Extract

Load the clarified extract onto the chitin column at a flow rate no faster than 0.5-1 ml/min. Take a sample of the flow through (sample 4) and compare it to the clarified cell extract sample to indicate the binding efficiency of the fusion precursor to the chitin column. If some of the fusion precursor is present in the flow through you may need to increase the amount of resin or load more slowly.

Washing the Chitin Column

At least 20 bed volumes of the Column Buffer should be used to wash the column (sample 5). Due to the high affinity of the CBD for the chitin beads, a higher flow rate (e.g., 2 ml/min) and stringent wash conditions can be used to reduce nonspecific binding of other E. coli proteins [high salt concentration (0.5-1 M NaCl) and/or nonionic detergents].

Induction of On-column Cleavage

To release the target protein, on-column cleavage is induced by a thiol reagent. Induction of the on-column cleavage is conducted by quickly flushing the column with 3 bed volumes of the Cleavage Buffer, containing 50 mM DTT, to evenly distribute thiols throughout the column (sample 7). After the quick flush, stop the column flow and leave at 4-23°C for 16-40 hours (see Tables 1A and 1B). Before adding the thiol reagent, check cleavage efficiency by removing 100 &mul of resin and mixing with 50 &mul 3X SDS Sample Buffer. After boiling for 5 minutes, spin the resin down. The supernatant (3-10 &mul) is directly used for SDS-PAGE analyses (sample 6).

If intein mediated protein ligation (IPL) or expressed protein ligation (EPL) is to be conducted, typically 2-mercaptoethanesulfonic acid (MESNA) is used as the thiol reagent to induce cleavage.

Several factors affect the cleavage efficiency and thus the final yield: (i) amino acid residue(s) at the cleavage site (ii) temperature of the cleavage reaction (iii) duration of the cleavage reaction (iv) pH of the Cleavage Buffer.

Since cleavage is dependent on the protein structure, a single amino acid residue at the cleavage site is not the only determinant for efficient cleavage, and only serves as a guideline. In most cases (see Tables 1A & 1B), incubation of the column at 16-23°C for 16 hours (overnight) results in more than 50% cleavage of the fusion precursor. When the C-terminal fusion vector (pTXB1) is used, the on-column cleavage reaction can be conducted at 4°C overnight. When the N-terminal fusion vector (pTYB21) is used, higher temperatures (16-23°C) and longer cleavage reaction times (40 hours) are normally required. The data in Tables 1A & 1B provides a guideline for selecting an appropriate temperature and duration for the cleavage reaction. The cleavage efficiency can be determined by a SDS-PAGE by analyzing samples of the chitin resin after thiol cleavage (sample 9).

If most of the precursor is not cleaved, longer incubation time and higher temperature for the cleavage reaction are recommended.

Elution of the Target Protein

Following on-column cleavage the target protein is eluted from the column using the Column Buffer. The intein-CBD tag remains bound to the resin. Fraction sizes of about one third of the column bed volume typically result in the elution of the target protein within the first few fractions (sample 8).

The protein concentration in each fraction can be determined by the Bradford Assay and the eluted fractions should be analyzed by SDS-PAGE. To check cleavage efficiency, remove 100 &mul of resin and mix with 50 &mul 3X SDS Sample Buffer. Boil for 5 minutes and spin the resin down. Analyze the supernatant (3-10 &mul, sample 9) by SDS-PAGE. If a large amount of the precursor still remains uncleaved, continue incubation of the column for an additional 12-24 hours before conducting a second elution.

When pTYB21 is used, a small peptide (1.6 kDa) is also cleaved from the intein tag and co-eluted with the target protein. Due to its small molecular weight, the cleaved peptide cannot be detected on a regular SDS-PAGE gel and can be removed by dialysis.

Stripping the Chitin Column

Uncleaved fusion precursor protein and the intein-tag remain bound to the chitin resin during elution and can be stripped from the resin by 1% SDS or 0.3 M NaOH in column buffer. The elution should be conducted at room temperature to prevent the precipitation of the SDS. Since protein concentrations cannot be determined by the Bradford dye binding assay when SDS is present, the samples should be analyzed by SDS-PAGE.

Regeneration of the Chitin Resin

The chitin resin can be regenerated 4-5 times using the following protocol. Wash with 3 bed volumes of 0.3 M NaOH (stripping solution). Allow the resin to soak for 30 minutes and then wash with an additional 7 bed volumes of Stripping Solution. Rinse with 20 bed volumes of water followed by 5 bed volumes of Column Buffer. The resin can be stored at 4°C. For long term storage 0.02% sodium azide should be added to the Column Buffer.

Note: Boiling in SDS Sample Buffer containing DTT can cause partial or complete cleavage, resulting in an overestimation of in vivo cleavage. If substantial in vivo cleavage is observed, the cell extract should be evaluated in a SDS Sample Buffer containing no DTT.

50% in vivo cleavagewhen induced at 15°C at 37°C in vivo cleavage was less than 5%.

50% in vivo cleavage when expression was induced at 15°C and 37°C.


Ilovalar

Most protein tags can be used for protein purification, and nearly all of them are suitable for protein detection using such techniques as Western blotting, ELISAs, immunohistochemistry, immunocytochemistry, measurement of fluorescence intensity, and ChIP-Seq. Certain tags have proven useful for solving challenging protein expression problems, e.g., by making it possible to express proteins that have lethal effects on host cells or protecting expressed proteins from proteolytic degradation (Li 2011). In addition, some tags may be used to help localize a target protein to a specific cellular compartment, or, as in the case of GST tags, to increase protein solubility.

Protein tags are most frequently used to purify proteins for which no protein-specific antibody exists. Such tags include his (polyhistidine), FLAG (DYKDDDDK), GST, and Myc tags, which are fused to proteins of interest using expression vector systems. Tag-specific capture reagents such as affinity resins or antibody-linked beads are available to purify proteins expressing these tags.

Ma'lumotnomalar

Li, Y. Recombinant production of antimicrobial peptides in Escherichia coli: A review. Protein Expr. Purif. 80, 260&ndash267 (2011).


GST-Tagged Protein Purification

G-Biosciences provide several reagents for affinity purification of glutathione S-transferase (GST) tagged proteins. The resin consists of reduced glutathione (GSH) covalently coupled to 6% cross-linked agarose, via a 10-carbon spacer arm. The resin has a binding capacity of

40mg GST/ml resin. Supplied as slurry in 20% ethanol. Glutathione Resin is available as resin alone or supplied in a kit format. Binding/ Wash and Elution buffers are available, in addition to reduced glutathione for purification.

For the purification of soluble GST-tagged proteins from bacterial cultures we provide HOOK&trade GST Protein Purification Kit (Bacteria). The bacteria are first lysed with Bacterial PE LB&trade and PE LB&trade-Lysozyme to release total soluble protein, whilst maintaining the structure and activity of the protein. The GST tagged protein is purified by passing clarified lysate through prepacked columns.

For the purification of soluble GST-tagged proteins from yeast cultures we provide HOOK&trade GST Protein Purification Kit (Yeast). The yeast are first lysed with Yeast PE LB&trade and LongLife&trade Zymolyase® to release total soluble protein, whilst maintaining the structure and activity of the protein. The GST tagged protein is purified by passing the clarified lysate through prepacked columns.

Rapid purification of soluble GST-tagged proteins from Bacteria or Yeast is simple and affordable with a HOOK&trade GST Protein Spin Purification Kit (Bacteria) and the HOOK&trade GST Protein Spin Purification Kit (Yeast). After lysis and release of the solubilized protein, the GST tagged protein is purified by affinity chromatography by adding 0.5ml glutathione resin to the clarified lysate. The resin is transferred to a convenient spin column, where it is rapidly washed and the GST protein is eluted.


Refolding of the solubilised proteins

Refolding of the solubilised proteins is initiated by the removal of the denaturant. The efficiency of refolding depends on the competition between correct folding and aggregation. To slow down the aggreagtion process refolding is usually carried out at low protein concentrations, in the range of 10-100 m g/ml. Furthermore, refolding conditions must be optimized for each individual protein. Important variables are:

FoldIt, a commercial protein folding screen is available from Hampton Research.

If proteins contain disulfide bonds, the refolding buffer has to be supplemented with a redox system. The addition of a mixture of reduced and oxidized forms (1-3 mM reduced thiol and a 5:1 to 1:1 ratio of reduced to oxidixed thiol) of low molecular weight thiol reagent usually provides the appropriate redox potential to allow formation and reshuffling of disulfide bonds. The most commonly used redox shuffling reagents are reduced and oxidized glutathione, but also cysteine and cysteamine are used.
For certain protein, probably due to low solubility of folding intermediates, this procedure is not very effective. Alternatively, the protein is completely oxidized in the presence of a large excess of oxidized glutathione, followed by dilution in refolding buffer containing catalytic amounts of reduced glutathione.

Different methods for the refolding of proteins have been described:

Dialysis. The most used method is the removal of the solubilizing agent by dialysis. During dialysis the concentration of the solubilizing agent decreases slowly which allows the protein to refold optimally. The ratio of the volumes of the sample and the dialysis buffer should be as such that at the equilibrium concentration of the solubilizing agent the protein has completely refolded.

Slow dilution. The concentration of the solubilizing agent is decreased by dilution allowing the protein to refold. Usually the dilution is carried out slowly by step-wise addition of buffer or by continuous addition using a pump.

Rapid dilution. During dialysis and slow dilution the protein is exposed for an extended period of time to an intermediate concentration of the solubilizing agent (2-4 M urea or guanidine-HCl) where it is not yet folded but no longer denatured and thus extremely prone to aggregation. This could be prevented by the rapidly dilution of the solubilized protein solution into the refolding buffer. Aggregation during this process can be limited by adding mild solubilizing agents to the refolding buffer, such as non-detergent sulfobetaines.

Pulse renaturation. In order to keep the concentration of the unfolded protein low, thus limiting aggregation, aliquots of denatured protein are added at defined time points to the refolding buffer. The time intervals between two pulses have to be optimized for each individual protein.The process is stopped when the concentration of denaturant reaches a critical level with respect to refolding of the specific protein.

Chromatography. The solubilizing agent is removed using a chromatographic step. The application of different chromatography methods have been described:

  • size exclusion chromatography (masalan. gel filtration on a Superdex 75 column)
  • ion exchange chromatography
  • affinity chromatography (masalan. IMAC using Chelating Sepharose or Ni-NTA agarose)

The denaturant is removed while the protein is slow migrating through the column or bound to the matrix. This usually gives a high yield of active protein even at protein concentrations in the mg/ml range.

Alternatively, it is possible to carry out chromatography under denaturing conditions before refolding the protein. Most modern chromatography resins are stable under the commonly used conditions for solubilization.


Videoni tomosha qiling: ХАР КУНИ ИККИТАДАН БАНАН ЕСАНГИЗ НИМА БУЛИШИНИ БИЛАСИЗМИ??? (Dekabr 2021).