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ImageJ Westernblot tahlilida yomon chiziqlarni qanday olib tashlash mumkin

ImageJ Westernblot tahlilida yomon chiziqlarni qanday olib tashlash mumkin


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Men ImageJ -dan Westernblot tasvirini tahlil qilish uchun foydalanaman.

Agar hamma narsa xohlagan tarzda bo'lsa, ish oqimi yaxshi bo'ladi. Agar men chiziq yaratishda noto'g'ri ish qilsam, chiziqni bekor qilish va men topadigan barcha qatorlarni olib tashlashning iloji yo'q.

Qanday qilib asl tasvirimga qaytishim yoki chiziq yaratishni bekor qilishim mumkin?

Men Mac OS da 1.49e dan foydalanmoqdaman


ImageJ-da alohida chiziqlarni olib tashlash xususiyati yo'q. Lekin bu muammo bo'lmasligi kerak. Siz faqat qilishingiz kerak birinchi qatorni to'g'ri chizish (Men o'lchamni nazarda tutyapman). Keyin bosing 1. Tanlash hali tugallanmagan bo'lsa -da, uning ichini bosing, lekin raqamni emas (kursor qo'lda bo'ladi) va uni keyingi bo'lak kerakli joyga torting. Va bosing 2. Va hokazo. Barcha chiziqlaringizni chizib oling.

Endi, agar siz yo'lni noto'g'ri joylashtirgan bo'lsangiz, to'rtburchaklar tanlash vositasini bosing, keyin kursor qo'lda bo'lsa, chiziq raqamini bosing. U tanlanadi. Agar sizda muammo bo'lsa, yorliqlardan foydalaning Ctrl+1 va Ctrl+2 yoki menyu Tahlil qiling - Jellar - Oldingi/Keyingi qatorni tanlang qatorlar orasidan tanlash uchun. Kursor normal holatga qaytadi. Yo'lak ichidagi biron bir joyni bosing va kerakli joyga torting.

Agar birinchi bo'lak yomon bo'lsa va siz barcha chiziqlarni olib tashlamoqchi bo'lsangiz, boring Tahlil qiling, Jeller va ustiga bosing Qayta o'rnatish.


Xolis tadqiqot

Molekulyar olim sifatida men ko'plab g'arbiy dog'larni bajaraman. Siz bilganingizdek, agar chiziqlar orasidagi farqni aniqlay olmasangiz, g'arbiy chiziqlar befoyda. Tasvir J (yoki FIJI) tez-tez g'arbiy dog'lardan olingan natijalarni aniqlash uchun ishlatiladi. Shu bilan birga, men har doim charchaganman, bu dastur bilan sizning miqdoriy fikringizga noaniqlikni kiritishning bir qancha usullari bor. Men hayron bo'ldimki, xuddi shu odam bir xil g'arbiy blotni bir necha marta tahlil qilsa, ular har safar bir xil qiymatlarga ega bo'larmidi? Buni o'rganish uchun men eski western blot tasviridan foydalanishga qaror qildim va qanday farqli qadriyatlarga ega bo'lishim mumkinligini bilib oldim.
Boshlash uchun men quyidagi g'arbiy dog'dan foydalandim. Bu shunchaki fitna mashg'uloti bo'lgani uchun, men faqat oxirgi uchta ustunning yuqori chiziqlarini tahlil qildim.

6 ta fikr:

Bu haqiqatan ham qiziqarli tahlil, men doim hayron bo'lardim. Guruh atrofidagi maydon tufayli urinishlaringiz o'rtasida farq borligi mantiqan to'g'ri keladi va men western blot tahlilini o'tkazayotganda buni hisobga olish mumkinmi, deb o'ylayman. Hududni tanlashning texnik nusxasi kabi bir narsa? Siz aytib o'tganingizdek, shovqinni to'g'irlash juda muhim va bu tafovutlar va potentsial tarafkashlikning yana bir qo'shimcha bosqichidir. Agar siz yukni boshqarish bilan bir xil tahlilni o'tkazgan bo'lsangiz va signalni normalizatsiya qilgan bo'lsangiz, men qiziqaman. Men tushundimki, bu shunchaki intriganing tezkor tajribasi edi va ehtimol bu yuqori guruh boshqaruvchi yoki soxta guruhdir. Nima bo'lishidan qat'iy nazar, sizning tahlilingiz ushbu turdagi tahlillarning oqibatlarini ko'rsatishi mumkin va u qiladi
Men yuklash nazorati yordam beradimi, deb o'ylayman. Yoki bu faqat hududni tanlash tarafkashlikning yana bir qatlami bo'lsa.
Bu shuni ko'rsatadiki, ba'zida biz ushbu guruhlarning intensivligining "statistik ahamiyati" ga ishonamiz. Shunga qaramay, bu faqat ilmiy aloqani bilmasdan (yoki umuman tajribani) bu guruhni kuzatganim, lekin men bu guruhlarga qarasam, intensivlik farqini ko'rmayapman. Sizning tahlilingiz shuni ko'rsatadiki, bu farqning biologik ahamiyatga ega ekanligini bilish juda muhimdir.

Men bu postni juda qiziq deb bilaman, mening laboratoriyam fiziologiya laboratoriyasi va agar kerak bo'lmasa, western blotlarini qilmaslikka harakat qilamiz. Yaqinda biz er -xotinni bajarishga majbur bo'ldik (har doim sharhlovchilarni xursand qilishga harakat qilardim) va men ImajeJ yordamida ma'lumotlarni tahlil qilar ekanman, xuddi shu narsani hayron qila boshladim. Rasmga tushganingizdan so'ng chiziqlar parallel bo'lsa ham, siz shovqinni, jelga yuklangan protein miqdorini va siz miqdorni aniqlash uchun to'g'ri maydonni tanlash uchun javobgar bo'lganingizda paydo bo'lgan noto'g'rilikni hisobga olishingiz kerak. Protein miqdoridagi farqlarni ba'zan dasturiy ta'minot yordamida yalang'och ko'z bilan aniqlash mumkin bo'lsa-da, biz kiritgan noto'g'riligimizdan xabardor bo'lishimiz kerak.
Amanda aytganidek, barcha miqdorlar mohiyat va sehrli p & lt0.05 ga qaytadi! Biroq, ko'rish ishonish mumkin, shuning uchun biz sizning 1 -chizig'ingiz 6 -qatorga qaraganda ingichka tasmaga ega ekanligini ko'rganimizda, buni eksperimental ahamiyatga ega deb qabul qila olamizmi? Yoki biz har doim ahamiyat paradigmasida qolamizmi?

Bu men uchun haqiqatan ham qiziqarli xabar, chunki men buni anchadan beri o'ylab yurganman, men Western Blot -ni birinchi marta qilganimda va guruhlarni tahlil qilish uchun ImageJ -dan foydalanishim mumkinligini aytishdi. Aslida, men so'nggi bir necha yil ichida juda ko'p nuqta qo'yganman, lekin ImageJ -dan haqiqatan ham foydalanganimda va intensivlikdagi farqni ko'rishni xohlaganimda. Bu hech qanday yordam bermaydi degani emas, lekin men guruhning intensivligini ehtiyotkorlik bilan tahlil qilish uchun ImageJ -dan foydalanishni tavsiya qilaman.

ImageJ tahlilini qiyinlashtiradigan bir necha omillar mavjud. Birinchi narsa - siz ishlab chiqarayotgan reaktiv va filmlarning sezgirligi yoki siz foydalanadigan tasvirlash tizimi. Tashqarida bir nechta tanlov mavjud va biz laboratoriyada an'anaviy kino tizimidan foydalanamiz va u ozgina farqni sezmaydi. Aslida bizning laboratoriya a'zolarimizdan biri yuklash boshqaruvini sinab ko'rdi, u erda xuddi shu oqsil lizatlarini skaler miqdorida yukladi, aytaylik, 2ug, 4ug, 8ug, 20ug. Keyin u dog'ni amalga oshirdi va ImageJ bilan tahlil qildi va u birinchi navbatda yuklaganidek, intensivlik o'sishini ko'rmadi. Trendlar to'g'ri edi, ko'proq yuklanish bilan, albatta, katta intensivlik, lekin u 2ug yuklashga qaraganda 4ugda ikki barobar yoki 8ugda 4 barobar emas edi. Shunday qilib, men laboratoriya sharoitida statistik tahlilni o'tkazishdan tashqari, ImageJ intensivligini tahlil qilish natijalariga shubha qilaman.

Yodda tutadigan ikkinchi narsa shundaki, agar sizda juda sezgir tizim bo'lsa ham, siz xato yoki noxolislik ehtimolini kamaytirish uchun siz solishtirgan narsalar bitta tajribada bajarilganiga va bir martalik tahlil qilinganligiga ishonch hosil qilishingiz kerak. Siz egri ostidagi qiymatni hisoblash uchun pastda chiziqni belgilash haqida blogda ko'rsatganingizdek, bu juda murakkab bo'lishi mumkin, chunki siz har xil tahlil davrida turli xil chiziqlarni chizishingiz mumkin. O'ylaymanki, bu bizning sinfimizdagi bitta ma'ruza bilan bog'liq bo'lib, siz avval barcha kerakli ma'lumotlarni to'plashingiz kerak, keyin esa reja bo'yicha o'tirib tahlil qilishingiz kerak.

Shu bilan birga, maslahatchim har doim shunday deydi: "ma'lumotlarni tahlil qilish va statistikani ko'rsatish uchun imageJ dan foydalanmang, avvalo meni ko'z qorachig'im orqali ishontirishingiz kerak"

ImageJ bilan ishlashning asosiy isboti uchun rahmat. O'ylaymanki, siz o'lchovlarni eksperimental tahlil qilish uchun ishlatishdan oldin ularning to'g'riligini aniqlashingiz va ulardan xulosa chiqarishingiz kerak. Bu tasdiqlar har doim nashrdan keyin o'quvchi uchun ochiq emas va men boshqa texnika bilan standartlashtirish va miqdorlarni aniqlash haqida hayron bo'ldim. Yaqinda bizning dasturimizda seminar ma'ruzachisi shtammlarning bog'liqligiga qarab agar plastinkalarida bir -biriga o'ralgan zonalarini birlashtiradigan yoki birlashtirmaydigan bakteriyalar haqida gapirib berdi. & Quot; Birlashtirish & quot; xulq -atvori ikki to'da zonasi o'rtasida & quot; chegara chizig'i & quot; tashkil etilganligi bilan belgilanadi. Chegara chiziqlarining ba'zi tasvirlari noaniq edi va farqlar mutlaq ko'rinmasdi. Bu erda ImageJ -ni tasdiqlaganingizdek, aholining xatti -harakatlarini aniqlashning ishonchli va aniq usulini yaratish, mening ma'ruzachi ma'lumotlari va xulosalariga bo'lgan ishonchimni kuchaytirgan bo'lardi. Umid qilamanki, biz olimlar sifatida rivojlanayotganimizda, biz sizning usullarimizni tasdiqlash va xulosalar haqida ko'proq ma'lumotga ega bo'lish orqali ilmiy yaxlitlikka intilamiz.

G'arbiy blotlarning ImageJ miqdorini aniqlashda men asosiy muammoga aylandim, shovqinni olib tashlash uchun chiziqni qaerga chizish kerak. Menga cho'qqining bir tarafidagi platodan (boshlang'ich/fon signalini ifodalovchi) boshqa tomondan platoga chiziq chizish o'rgatilgan, ma'lumotni tahlil qilayotgan boshqa odamga esa mahalliy minimalardan chiziq chizishni o'rgatishgan. tepalikning narigi tomonidagi mahalliy minimalarga. Biz ba'zan juda boshqacha natijalarga erishardik, shuning uchun mening ballim va uning balli o'rtasida farq bor yoki yo'qligini bilish uchun bir nechta g'arbiy mamlakatlarimizdagi ballar uchun ikkita t-testini o'tkazishga qaror qildik. Biz borligini aniqladik va oxir -oqibat, biz to'plash usullarini standartlashtirishga majbur bo'ldik. Biz uchrashishimiz mumkin bo'lgan ko'p xilma -xillik va sharoitlarni o'z ichiga olishga harakat qilish uchun protokol ishlab chiqdik va natijada bizning baholarimiz deyarli farq qilmadi. Biroq, ushbu protokolni yaratish jarayonida biz g'arbiy tomondan muntazam ravishda yuguradigan bir nechta postdocs va kichik PIlar bilan gaplashdik va ularning har birida bu tahlilni qanday bajarish kerakligi borasida o'z farqlari bor edi. Men G'arbni ImageJ tahlil qilish bo'yicha rasmiy protokoldan xabardor emasman, lekin kamdan -kam hollarda (agar bo'lsa ham) bu tahlil qanday o'tkazilganligi (agar ular ImageJ yordamida miqdoriy tahlil o'tkazgan bo'lsalar) va to'purarlar qanchalik o'zgarishi mumkinligini hisobga olgan holda. ularning usullari, ehtimol, yanada rasmiy protokol zarur. Agar ma'lumotlarning o'zi noto'g'ri bo'lsa, hech qanday statistik ma'lumot buni to'liq tuzatolmaydi.
G'arbiy ma'lumotlarga ko'ra, bizning laboratoriyamiz shunday yo'l tutganki, biz natijani sifat jihatidan ko'ra olmasak, g'arbiy qismida miqdoriy tahlil qilish juda muhim (ya'ni, bu guruhni ko'rish imkoni bor edi). o'lchamlar yalang'och ko'z bilan farq qiladi), ayniqsa bizning tahlil usullarimizdagi o'zgaruvchanlik darajasini hisobga olsak, sizning natijangiz ishonarli emas va ehtimol unchalik katta bo'lmagan tuz donasi bilan qabul qilinishi kerak va shuning uchun nashr etilishi mumkin emas. G'arbda ishlaydigan sizlarning laboratoriyalaringiz sifat va miqdoriy tahlillarga nisbatan qanday tajribaga ega ekanligini eshitish qiziq bo'lardi.

G'arbliklar mening bakalavriyatimda non va sariyog'im edi va hatto o'sha paytda ham ularning miqdorini aniqlash meni asabiylashtirdi, shuning uchun oqim syujetlari hozir ham yo'q. Siz har doim shovqin darajasini ko'rdingiz, deyish uchun juda halol usullar postini his qilasiz. G'arbliklar ham meni har doim tashvishlantirardilar, chunki ma'lum bir nuqtadan so'ng, siz yuqori signalni yo'qotasiz, chunki film iloji boricha ishlab chiqilgan va hozirda ochilgan qismi uning atrofidagi maydonni cheklab qo'yishi mumkin, u ham reaksiyaga kirisha oladi. G'arbliklar hali ham ajoyib vosita, lekin men xohlaymanki, biz ularni talqin qilish uchun to'g'ridan -to'g'ri miqdoriy usullarni ishlatmagan bo'lardik, yoki ularning mavjudligiga/yo'qligiga yoki qonli aniq farqlarga qaraganda kamroq ishonardik.


Western blotning tarmoqli intensivligini qanday aniqlash mumkin? - Ilmiy ko'rgazma va#33 uchun yordamga muhtoj o'rta maktab o'quvchisi (2005 yil 03 fevral)

Men ilmiy ko'rgazma loyiham uchun Western Blot yordamida tajriba o'tkazyapman.

Grafik yaratish uchun adobe Photoshop 5.5 yoki 7.0 yordamida chiziqlar intensivligini baholash kerakligini eshitdim. Menga kimdir aytishi mumkinmi, deb o'ylardim, iltimos, dastur yordamida hosil qilingan guruhlarning piksellarini/intensivligini qanday olish kerak?

Rahmat ! Men yordamni qadrlayman !

Men buni fotoshop yordamida qilish mumkinligini eshitganman, lekin bu borada tajribam yo'q. laboratoriyamizda Scion Corp.dan ScionImage -dan foydalanamiz.

Buni shu yerda olishingiz mumkin, garchi qo'llab-quvvatlash faqat pullik mijozlarga beriladi.

lekin keyin, ehtimol, kimdir PS dan miqdoriy aniqlash uchun foydalanadi va sizga yordam berishi mumkin.

salom
Men sizga protsedurani aytmayman, lekin miqdorni aniqlash sifatini ta'kidlamoqchiman. Ochig'ini aytganda, men eshitgan yagona usul - bu sizning ekstraktlaringizni va har xil molekulyar og'irlikdagi turli xil oqsillarga ega bo'lgan ma'lum miqdordagi 2, 4, 6, 8 va hokazo oqsillarni yuklashni o'z ichiga oladi va agar siz antikorlardan foydalansangiz, filmni ochib beradi. bir xil ta'sir qilish vaqti bilan (amalda bu asosiy vahiyni aytadi). Agar siz taxminiy (lekin unchalik yomon emas) qiymatni istasangiz, jadefalcon aytganidek, fotoshopdan foydalanishingiz mumkin.

Siz qilishingiz kerak bo'lgan birinchi narsa - jelingizning yaxshi ta'sirini olishdir. Bu shuni anglatadiki, siz haddan tashqari ta'sir qilmaysiz. Qoida tariqasida, siz bantlarni ko'rishingiz kerak (ya'ni uni qog'ozga qo'ying va u orqali matnni o'qiy olasizmi yoki yo'qmi).

Ikkinchidan, siz solishtirmoqchi bo'lgan BARCHA guruhlar bir xil filmda bo'lishi kerak. Miqdori 1 (yoki 100%) deb qo'ygan bitta bandga nisbatan.

1) PS-da sizga rasmingizni yuklang
2) Keyin qiling, Image & gtAdjust & gtInvert (shu tariqa siz o'lchovni teskari aylantirasiz)
3) Marquee Tool yordamida ONE bandini qamrab oluvchi qutini chizib oling (birinchi navbatda eng katta bandni bajaring, chunki bu tahlil butun o'lchovda bir xil bo'lishi kerak. Shunday qilib, qutining hajmini hisobga olmaslik kerak). )
4) Keyin, Image>Histogram-ni bajaring va O'rtachani yozing
5) Keyin qutini keyingi bandga torting va takrorlang
6) Nihoyat, fondan o'rtachani o'lchash uchun katakchani torting va qolgan barcha raqamlardan raqamni olib tashlang.
7) Barcha raqamlarni mos yozuvlar raqamingiz bilan bitta to'plamga bo'ling (yoki 100%).

Buni amalga oshirishning juda qo'pol usuli, lekin sizga ifodaning nisbiy oshishi/kamayishi haqida ba'zi maslahatlar bering.

Shuni esda tutingki, skaner yordamida filmingizning raqamli tasvirini yaratganingizda, iloji boricha yuqori sifatli tasvirni yaratishingiz kerak. Biz filmlarimizni kulrang rangda skanerlaymiz va 1200 dpi aniqlikdagi TIFF faylini chiqaramiz.

Filmni jpeg qilmang, jpeg -da tasvir ma'lumotlari tiff fayllariga qaraganda ancha kam.

Yuqoridagi Scion tasvirini ishlatib ko'ring. Guruhlar miqdorini aniqlash uchun fotoshopdan foydalanish tajribam yo'q.

Tiff faylini Scion tasviriga yuklang. Men hech qachon skion tasvirini ishlatmaganman, lekin bu printsip har qanday tasvir dasturida bir xil bo'ladi deb o'ylayman. Ishonchim komilki, bu usul mukammal emas, lekin u siz uchun ishlashi mumkin

Birinchidan, tasmasi bo'lmagan hududga qutichani torting va tahlil-& o'lchovni tanlang. Bu sizga tasviringizning fon intensivligini (o'rtacha), shuningdek, qutingizning maydonini aytib beradi, bu raqamlarni yozing.

Keyin, o'lchamoqchi bo'lgan diapazon atrofida ROI (qiziqish hududi) ni chizing va tahlil qilish-> o'lchovini tanlang. Siz yana maydon o'lchovini va o'rtacha intensivlikni olasiz. Bu raqamlarni yozing. Buni o'lchashni xohlagan barcha chiziqlar uchun takrorlang.

O'zingizning ROI -laringizni va men ' ROI -ni eksperiment qilishda ehtiyot bo'ling va siz ularni to'g'ri chizish va aniq o'lchash mumkin deb o'ylamaguningizcha, ulardan olingan qiymatlarni solishtiring.

Men xato qilgandirman, lekin sizning guruhlaringizni hisoblash uchun siz kerakli sonni olish uchun o'rtacha intensivlik va maydonni ko'paytirishingiz kerak bo'ladi. Buni sizning foningiz uchun ham qiling. Bu raqamlarni yozing.

Keyin har bir tasma qiymatidan fon qiymatini (o'rtacha maydon intensivligi x) olib tashlang (x maydon o'rtacha zichlik)

Agar bu mantiqiy bo'lsa, menga xabar bering. Bundan tashqari, kimdir mening tasvirlangan usulimni yaxshilashi mumkin bo'lsa, buni qilishdan tortinmang

Men chiziqlar intensivligini qanday aniqlashni bilmayman, lekin Internetdan yuklab olish mumkin bo'lgan ImageJ (NIH dan) deb nomlangan bepul dasturiy ta'minot mavjud bo'lib, bizning laboratoriyamiz G'arbiy dog'larimizning densitometriya tahlilini o'tkazish uchun foydalanadi.

Nega aniq manzilni bermaysiz.

farrux
Iltimos, oxirgi xabarning sanasiga e'tibor bering

Agar siz Google'dan foydalansangiz, manzilni topishingiz mumkin deb o'ylayman? yoki, muqobil, NIH saytini qidirganmi?

Yana biri western blot miqdori va Scion Image (yoki ImageJ) bo'yicha…

Mana, men olgan natijalarning tasviri:

Rasmda ko'rsatilgandek natijalarga ega bo'lsak, uning intensivligini emas, balki guruhning hajmini (maydonini) ham hisobga olish yaxshiroq bo'larmidi?

Menda Scion Image -dan foydalanish tajribasi juda kam, lekin Gelplot makrosidan foydalanganda, men faqat to'rtburchaklar tanlash vositasi bilan turli yo'llarni tanlashim mumkin va to'rtburchaklar tanlovining o'lchami har bir bo'lak uchun bir xil bo'lishi kerak.

Qiziqarli joyni chizish o'rniga, men sehrli tayoqchani tanlashni xohlardim, lekin bu rasmning ostonasini nazarda tutadi, bu yaxshi fikrmi?

O'lchovlar oldidan tasvirga har qanday modifikatsiyani (chegara/kontrastni oshirish/...) qo'llaysizmi?

Butun plyonka uchun bitta fon qiymatini olib tashlashning o'rniga (filmning istalgan joyidan olingan to'rtburchaklar tanlovdan), har bir bo'lakka fon qiymatini olib tashlash maqsadga muvofiqmi yoki vaqtni behuda sarflashmi (faqat yuqorisida yoki pastda). chiziq).


Kirish

Hujayralar markaziy dogma bo'yicha tartibga solinadi, bu genetik ma'lumotdan oqsil ekspresiyasiga o'tishdir. Proteinlarning ifoda darajasi hujayralar taqdirini belgilaydi, masalan, transkripsiyaning ba'zi omillarining skelet mushaklari differentsiatsiyasini tartibga soladi [1]. Shunday qilib, molekulyar darajada hujayra hodisalari yoki potentsialini tushunish uchun protein ifodasini miqdoriy aniqlash juda muhimdir. Western blotting - bu protein ifodasini aniqlashning standart usuli [2]. Xemiluminesans usuli odatda yuqori sezuvchanlik va foydalanish qulayligi tufayli oqsillarni aniqlash uchun ishlatiladi [3, 4]. Shu bilan birga, proteomikaning so'nggi rivojlanishi bir xil oqsillarni aniqlaydigan xemiluminesans usulidan farqli o'laroq, bir vaqtning o'zida bir nechta oqsillarning ifodasini aniqlash texnologiyasiga qaratildi. Bir vaqtning o'zida bir nechta oqsillarni aniqlashning ko'proq miqdoriy tahlil qilish usullaridan biri floresan g'arbiy blotdir [5-7]. Biroq, bu usulning aniqlash sezgirligi kimilyuminesans usulidan pastroq.

Ushbu tadqiqotda floresan g'arbiy blotning o'lchamlari va sezgirligini yaxshilash uchun biz flüoresan mikroskopiya bosqichiga e'tibor qaratdik. Mikroskopning CCD kamerasi yuqori aniqlikdagi tasvirlarni olishga qodir, lekin u kichik ko'rish maydoniga ega. Shunday qilib, biz bir nechta lyuminestsent mikrograflarni birlashtirib, yuqori aniqlikdagi va keng maydonli tasvirni oldik. Bundan tashqari, biz filtrlarni va lyuminestsent bo'yoqlarni optimallashtirish orqali aniqlash sezuvchanligini xemiluminesans usuli bilan solishtiradigan darajada muvaffaqiyatli oshirdik.


Avtofloresansni olib tashlash usullari

Agar siz avtofloresans bilan bog'liq muammolarga duch kelsangiz, birinchi navbatda, sizning namunangizni o'rganib, sababini ajratib ko'rsatish kerak. Tajribalarni boshlaganingizda, bo'yash jarayoni fon floresansiga hissa qo'shmasligini aniqlash uchun yorliqsiz boshqaruvni ishga tushiring.

Avtofloresans spektrini bilish ham muhimdir. Buni spektral lambda ko'rish yordamida aniqlash mumkin. Spektral skanerlash eksperimentni optimallashtirishga va avtofloresansning kuchli cho'qqilaridan qochishga yordam beradi.

Avtofloresans spektrlari haqida yaxshi tasavvurga ega bo'lganingizdan so'ng, keyingi bosqich - florofor tanlovlarini optimallashtirish. Agar avtofloresans spektrlari va sizning floroforingiz bir -biriga juda mos kelsa, sizning signalingiz avtofloresans yordamida yashirilishi ehtimoli katta. Bunday holda, sizning fon avtofloresansidan uzoqda joylashgan spektrli floroforni tanlang. Misol uchun, agar sizning avtofloresaningiz spektrning ko'k mintaqasida bo'lsa, floroforingizni yashil yoki qizil rangga o'tkazing.

Ftoroforlarni tanlashda Alexa Fluor, Dylight yoki Atto kabi zamonaviy probni tanlang. Bu bo'yoqlar odatda yorqinroq, barqarorroq va qo'zg'aluvchanlik va emissiya diapazonlari torroq bo'lib, ftoroforingizdan faqat signalni tanlashni osonlashtiradi. Agar mikroskopingiz ularni aniqlay olsa, uzoq qizil bo'yoqlar avtofluoresans bilan bog'liq muammolarni oldini olishning juda yaxshi usuli hisoblanadi, chunki bu to'lqin uzunliklari biologik namunalarda kamdan-kam uchraydi. Uzoq qizil bo'yoqlarni aniqlash qobiliyati ko'p rangli tajribalar uchun ishlatilishi mumkin bo'lgan kanallar sonini kengaytirish imkoniyatiga ega.

Tanlangan florofor bilan, kolbada ko'rsatilgan konsentratsiyalar bilan tajribangizni ko'r-ko'rona o'tkazmaslik muhimdir. Ko'pincha sizning namunangizda turli konsentratsiyalarni sinab ko'rish uchun ftor ftorini titrlash kerak bo'ladi. Bu sizga qaysi kontsentratsiyalar sizning foningizdan eng yaxshi farqlanishini va eng past avtofloresansni berishini ko'rishga imkon beradi. Qo'llanma sifatida ishlab chiqaruvchining ko'rsatmalaridan foydalaning va tavsiya etilgan foydalanishdan bir oz pastroq va biroz yuqoriroq oraliqni qamrab oladigan ftoroforni suyultirish seriyasini yarating. O'zingizning namunangizga nisbatan bu suyultirishlarni sinab ko'ring, bu sizga eng foydali signalni beradi.

Mikroskop sozlamalarini optimallashtiring

Konfokal mikroskopingiz sozlamalari sizning rasmingizda signallar, shu jumladan avtofloresans ko'rinadigan katta rol o'ynaydi. Spektral aniqlashni moslashtirish orqali avtofluoresans signalini imkon qadar ko'proq kesish uchun ushbu sozlamalardan o'zingizning foydangiz uchun foydalaning. Mikroskopga qarab buni qilishning turli usullari mavjud, lekin eng moslashuvchan variant - oq yorug'lik lazerini spektral detektor bilan birlashtirish. Bu sizning namunangizga keladigan yorug'lik to'lqin uzunliklarini, shuningdek detektordan o'tadigan to'lqin uzunliklarini aniq sozlashingiz mumkinligini anglatadi. Bu tasvirga qaysi signallar yozilishini tanlashda juda nozik nazorat qilish imkonini beradi va avtofloresansni yo'q qilish uchun keng imkoniyat beradi.

1 -rasm: Avtofloresans va florofor spektrlarini solishtirganda, avtofloresans signaliga to'g'ri kelmaydigan ftoroforni tanlang.

Avtofluoresansni oldini olish uchun namunangizni davolash

Avtofluoresans ko'pincha namunadan emas, balki tasvirlashdan oldin uni davolash usulidan kelib chiqadi. Masalan, o'rnatish vositasi, to'qima madaniyati muhiti va laboratoriya plastmassasi avtofloresans manbai bo'lishi mumkin.

Agar siz jonli hujayrali tasvirlash tajribalarini o'tkazayotgan bo'lsangiz, tasvirlashdan oldin oddiy madaniyat muhitini oldindan qizdirilgan fenol qizilsiz muhit yoki shaffof buferli tuz eritmasi bilan almashtirishni o'ylab ko'ring. PH indikatori fenol qizilidan tashqari, u 440 nm tezlikda qo'zg'alganda yuqori darajada lyuminestsent bo'ladi, madaniy muhit va FBS kabi qo'shimchalarda ko'plab oqsillar va o'z -o'zidan lyuminestsent signalli kichik molekulalar bo'lishi mumkin, bularning barchasi kuchli avtofloresans fonini qo'shishi mumkin agar ular olib tashlanmasa. Agar siz hujayralarni jonli tasvirlash uchun yangi muhitga o'tkazishga qaror qilsangiz, shuni bilishingiz kerakki, bu hujayra xatti -harakati va fenotipida kutilmagan o'zgarishlarga olib kelishi mumkin, shuning uchun siz o'rganayotgan narsangizga qarab hujayralarni moslashtirishingiz kerak bo'lishi mumkin. birinchi navbatda yangi vosita.

Xuddi shu mikroskop yordamida siz ishlatayotgan tasvir idishlaridan avtofloresansni o'lchashga arziydi, bu avtofloresans manbai yoki yo'qligini bilish uchun. Agar shunday bo'lsa, olib tashlash uchun maxsus mo'ljallangan shisha oynali madaniyatli idishlar yoki shisha taglikli idishlar yordamida tasvirlashga harakat qiling.
avtofloresan signallari.

Xuddi shunday muammolarni kimyoviy ta'sirga uchragan biopsiya va to'qima namunalarini tasvirlashda ham ko'rish mumkin. Umumiy misol - ovqat hazm qilish trakti, agar u kuchli lyuminestsent belgilarga ega bo'lgan tetratsiklin kabi antibiotiklarga ta'sir qilgan bo'lsa, katta miqdorda avtofluoresansga ega bo'lishi mumkin. Bunday holda, floresansni buferli sho'r suv bilan yaxshilab chayish yoki erituvchilarni ehtiyotkorlik bilan ishlatish orqali osonlikcha olib tashlash mumkin.

Agar siz hujayralarni tuzatayotgan bo'lsangiz, fiksaj usuli avtofloresansga katta ta'sir ko'rsatishi mumkin. Formalin va glutaraldegidga alternativalarni ko'rib chiqing va tasvirni olishdan oldin namunalarni uzoq vaqt saqlamaslikka harakat qiling, chunki vaqt o'tishi bilan avtofloresans kuchayishi mumkin.

Eksperimental dizayn orqali avtofluoresansni o'tkazib yuborish maqbul bo'lsa-da, so'm vaqtlarda bu mumkin emas. Bunday hollarda, avtofloresans bilan bog'liq muammolaringizni hisoblash echimlari bo'lishi mumkin. Mikroskopiya dasturidan yoki ImageJ kabi ochiq manbali echimlardan foydalanib, aut floresansni o'z ichiga olgan piksellarni tahlil qilish va uni umumiy tasvirdan olib tashlashga harakat qilish mumkin [1]. Ehtiyot bo'ling, hisoblash yondashuvlari murakkab bo'lishi mumkin. Tanlash uchun juda ko'p turli xil usullar va algoritmlar mavjud, shuning uchun qaysi biri yaxshi ishlashini ko'rish uchun bir nechtasini sinab ko'rish yaxshidir. Shuni esda tutish kerakki, bu usullar sizning florofor signalingizning kuchini, shuningdek, avtofloresansni kamaytirishi mumkin, shuning uchun ularni diqqat bilan ishlating va shuni bilingki, tez -tez tayanch bilan kontrastning oshishi va signal intensivligining pasayishi o'rtasida kelishmovchilik bo'ladi.

Fiksatsiyadan keyin avtofloresansni olib tashlash

Namunalaringizni tayyorlab, muzlatgichda saqlaganingizdan so'ng, har qanday avtofloresans qolishi kerakdek tuyuladi. Namunani tayyorlash bosqichida avtofloresansni yo'q qilish osonroq bo'lsa -da, jarayonning keyingi bosqichlarida ham amalga oshirilishi mumkin bo'lgan qadamlar bor.

Avtofloresans signallarini susaytiradigan ko'plab kimyoviy muolajalar mavjud. Ulardan ba'zilari sotuvda mavjud, boshqalari natriy borohidrid, Sudan qora B, ammiakli etanol va boshqalar kabi oddiy laboratoriya kimyoviy moddalari yordamida oson tayyorlanishi mumkin [2,3].

Photobleaching odatda mikroskopda salbiy ma'noga ega bo'ladi, bu uzoq vaqt lazer yordamida eng yaxshi tasvirni qidirishda floresan signalingizni yo'qotishi bilan bog'liq. Biroq, avtofloresans uchun oqartirish sizning do'stingiz bo'lishi mumkin. Ftoroforlarni qo'shishdan oldin, siz fonni avtofloresansni oqartirish uchun namunangizni yuqori zichlikdagi LED yorug'lik bilan davolashingiz mumkin. Shundan so'ng, siz tanlagan florofor oqartirilgan fonga nisbatan ancha kuchli kontrastga ega bo'lishi kerak [4].


NATIJALAR VA MUHOKAMA

Ushbu laboratoriya mashg'ulotini bajarish uchun 2 yoki 3 laboratoriya davri talab qilinadi (vaqt chizig'i uchun I jadvalga qarang). Birinchi kuni LB bulyonli suyuq kulturalari (2 ml) izolyatsiya qilingan koloniyalardan ilgari taralgan va o'qituvchi yordamchisi yoki o'qituvchi tomonidan inkubatsiya qilingan plastinkalarga ekiladi. Istalgan vaqtda (biz odatdagidek bajaradigan dastlabki emlash bilan birga yoki ma'lum bir o'sish vaqtidan keyin) madaniyatga induktor qo'shiladi (IPTG lak DH5a hujayralarida operon, HB101 hujayralarida pGLO plazmid uchun arabinoza) va inkubatsiya 37 ° C da chayqatish bilan davom etadi. Belgilangan inkubatsiya vaqtidan so'ng (kechaga 5 yoki 6 soat) 1,5 ml kultura mikrotsentrifuga naychasiga o'tkaziladi va hujayralar maksimal tezlikda 2 minut davomida santrifüjlash orqali pelletlanadi. 1 × SDS yuklash tamponida qayta suspenziya qilingandan so'ng, namunalar to'g'ridan-to'g'ri elektroforez uchun poliakrilamid jellarga yuklanishi yoki ishlatilgunga qadar -20 ° C da saqlanishi mumkin. Elektroforez va ishlov berish o'sha kuni amalga oshirilishi mumkin, lekin agar shunday bo'lsa, oqsillarni PVDF membranasiga o'tkazish ham o'sha kuni amalga oshirilishi kerak. Uzatilgandan so'ng, membranani 4 ° C da 1 yoki 2 kun davomida saqlash mumkin yoki uni to'g'ridan -to'g'ri blokirovka qilish bosqichiga o'tkazish mumkin, so'ngra birlamchi antikor bilan kechasi inkubatsiya qilish mumkin. Bularning barchasi bir vaqtning o'zida 3 soatga yaqin yoki 2 kunga bo'linishi mumkin. Yuvish, ikkilamchi antikor bilan inkubatsiya qilish va xemiluminestsent substrat yordamida rivojlanish 1 kun ichida bajarilishi kerak va 3 soat davom etishi kerak.

Tahlil qilish uchun odatiy talaba natijalari lak operon nazorati 3 -rasmda ko'rsatilganA. Osonlik bilan ko'rinib turganidek, IPTG induktori ishtirokida b-galaktosidaza oqsili uchun signalning ortishi kuzatiladi, ammo induktor yo'qligida signalning sezilarli miqdori mavjud. Bu farq qisqa vaqt ichida (10 soat bilan solishtirganda 5 yoki 6 soat) etishtirilgan namunalar uchun ko'proq seziladi. Bu farq umumiy oqsilning farqiga bog'liq emas, buni har bir bo'lakdagi oqsil miqdorini bir xil, Coomassie Blue bo'yalgan jelda solishtirish orqali ko'rish mumkin (3-rasm).B). Kechki madaniyatlar uchun induktsiyalangan va induktsiya qilingan protein namunalari o'rtasida b-galaktosidaza signalining farqi kamroq bo'ladi (4-rasm). Bu natijalar e'lon qilingan topilmalar bilan mos keladi, ular statsionar fazali madaniyatlarda nazorat kamayadi [5].

Bakterial hujayralardagi b-galaktosidaza ekspressiyasini Western blot tahlili. A, DH5a hujayralari "Materiallar va usullar" bo'limida ko'rsatilgan vaqtlarda IPTG induktori (+) yoki yo'qligi (-) mavjud bo'lganda inkubatsiya qilindi. pastki raqamdan. Umumiy oqsil qayta tiklandi va SDS-PAGE yordamida ajratildi, PVDF membranasiga o'tkazildi va sichqonchaga qarshi b-galaktosidaza antikorlari bilan inkubatsiya qilindi. Aniqlash sichqon-HRPga qarshi antikor yordamida xemiluminesans yordamida amalga oshirildi. B, Coomassie Blue-bo'yalgan jel, induktsiyalangan (+) va indüksiyalanmagan (-) hujayralardan jami oqsilni ko'rsatadi. Jel quyida ko'rsatilgan Western blot uchun ishlatiladigan jel bilan bir xil tarzda yuklangan A. M, marker oqsillari jel tarkibiga kiradi (Bio-Rad Precision bo'yalmagan markerlar, dan molekulyar og'irliklari bilan yuqori ga pastki 250, 150, 100, 75, 50, 37, 25, 20, 15 va 10 kDa).

Bir kechada (statsionar faza) madaniyatlarda b-galaktosidaza ifodasining g'arbiy bloti. Ko'rinib turibdiki, induktsiyalangan va induktsiyalangan protein namunalari o'rtasida farq kamroq.

Talabalar uchun arabinoza operonining g'arbiy dog'lari natijalari 5-rasmda ko'rsatilgan. Birinchi ikkita chiziqda operon uchun induktor bo'lgan arabinoza (+) va yo'qligi (-) bo'lganda o'stirilgan tungi madaniyatlarning hujayra lizatlaridan jami oqsillar mavjud. Blot GFPga qarshi poliklonal antikorlar bilan inkübe qilingan, bu konstruktsiyada arabinozani targ'ibotchisi bilan bog'langan muxbir genining mahsuloti. Induktsiya qilingan namuna uchun GFP o'lchami uchun kuchli signal kuzatiladi, aniqlanmagan bo'lakda esa aniqlanadigan signal kuzatilmaydi. Yuqori molekulyar og'irlik tasmasi-bu antikor bilan o'zaro reaksiyaga kiradigan, bir-biriga bog'liq bo'lmagan oqsil. Ba'zi namunalar GFPni konsentratsiyalash uchun immunopresipitatsiyaga uchradi, buni IP+ bo'lagida juda konsentrlangan GFP diapazoni kuzatishi mumkin (5 -rasm). Arabinoz induktorisiz etishtiriladigan bir kechada etishtirish uchun mos o'lchamdagi tasma aniqlanadi, lekin induksion chiziqli chiziq bilan solishtirganda unchalik kuchli emas. Faqat 5 soat davomida etishtirilmagan namunada guruh deyarli aniqlanmaydi. Namunalar orasidagi farqlar kuzatiladi va bu indüksiyaga qadar va undan keyin o'sish davomiyligidagi farqlar va inkubatsiya haroratidagi kichik farqlar natijasida bo'lishi mumkin. Inkubatsiyadan keyingi boshqa farqlar ham o'zgarishlarga olib kelishi mumkin, shu jumladan bakterial hujayralarning etarli darajada granulalanishi yoki qayta suspenziyasi, SDS-PAGE dan oldin oqsillarning to'liq denaturatsiyasi, jelni yuklashdagi xatolar, uzatish yoki o'tkazishdan keyingi bosqichlar.

Arabinoz-induktiv promouterdan GFP ifodasini Western blot tahlili. PGLO plazmidini tashuvchi HB101 shtammlari bir kechada arabinoz induktor ishtirokida (+) yoki yo'qligida ( -) o'stirildi, umumiy oqsillar avvalgidek izolyatsiya qilindi. chap panel, etiketli L lizat uchun) va namunalar SDS-PAGE orqali ajratilgan. Keyin oqsillar PVDF membranasiga o'tkazildi va "Materiallar va usullar" bo'limida ta'riflanganidek, quyon anti-GFP antikori bilan inkubatsiya qilindi. The o'ng panel IP kontsentratsiyasidan keyin ko'rsatilgandek, bir kechada yoki 5 soat davomida induktor ishtirokida (+) yoki yo'qligida ( -) o'stirilgan namunalarni o'z ichiga oladi. M Belgilangan oqsillarni o'z ichiga olgan marker chizig'i (30 va 40 kDa belgilarini kuzatish mumkin). The o'qlar ∼29-kDa GFP oqsil hajmini ko'rsating. Yuqori molekulyar og'irlikdagi bantlar, ko'rinishidan, hujayralardan o'zaro ta'sir qiluvchi artefakt tufayli.

Potentsial muammolar/muammolarni bartaraf etish

Weak signals may be due to one or more of the following: insufficient transfer from gel to membrane, incorrect transfer buffers, incubation with antibody was not long enough, or antibody is too dilute.

Too much background can result from using too much antibody or from allowing the membrane to dry out during the incubation steps. Enough solution should be used to keep the membrane completely submersed but without wasting excess solution and antibody. We have found that a pipette tip box lid is an ideal size for holding 20–30 ml of solution. The proper dilution of antibody needs to be empirically determined.

Suboptimal immunoprecipitation conditions may lead to reduced protein signal or an increase in nonspecific bands and background staining following Western analysis. Salt and detergent concentrations in the IP buffer can be adjusted as needed to maximize efficiency and specificity.

Questions for Classroom Discussion

What is the basis of any differences in the amount of β-galactosidase observed in Western blots relative to time of growth?

Does there appear to be more control of gene expression in the logarithmic stage of growth ga qarshi stationary phase?

Why should the time when inducer is added to the culture affect expression levels if the cells are grown to the same final density?

How might temperature of culture incubation affect expression what would be some possible reasons for altering the temperature?

Upon comparison, does the ara promoter show greater or lesser control over gene expression when compared with the lak promoter?

How does immunoprecipitation enhance detection levels of proteins?

When would lak promoter constructs have the advantage over ara promoter constructs?

For what applications would the ara promoter be most useful?

What modifications to either promoter might be useful to obtain optimal expression under specific growth conditions?


Natijalar va muhokama

Optimization of PCFT expression

In pilot studies where we varied the number of viral particles per cell (multiplicity of infection, MOI) and density of Sf9 cells grown in a 50-ml suspension culture, we found that PCFT expression was better at a MOI of 2 and a density of 2 x 10 6 cell/ml. Fig 1A shows PCFT expression under these conditions, as a function of time after infection. For each timed sample, we determined cell viability and PCFT expression by Western blot. PCFT expression peaked 48 h post-infection with a cell viability of

40%. In summary, the following conditions yielded the best PCFT expression: Sf9 cells infected at a density of 2 x 10 6 cell/ml with a MOI of 2 and harvested 48 h after infection.

A. Sf9 cells in 50 ml suspension culture at a density of 2 x 10 6 cells/ml were infected at a MOI of 2. One-ml whole cell samples were collected at the indicated time points, electrophoresed on a 4–15% Mini Protean TGX Precast SDS-PAGE gel (BioRad), transferred to a PVDF membrane, and immunoblotted using an antibody against the His6 tag of PCFT. PCFT bands were observed at

43 kDa. The highest PCFT expression was observed 48 h post infection. No PCFT expression was observed at the time of infection (0 h) or in uninfected cells after 48 h (uninf). B. Treatment of membrane vesicles with PNGase F under non-denaturing conditions shifted the PCFT band from

39 kDa (each lane treated with PNGase corresponds to a different sample preparation).

The whole cell samples used for the initial expression investigations yielded two close bands (

43 kDa) in Western blots. N-linked glycosylation of two asparagine residues within the first extracellular loop has been observed in recombinant human PCFT expressed in mammalian cells or Xenopus laevis oocytes [26, 31]. In these expression systems, treatment with PNGase F shifted the PCFT band in Western blots from

35 kDa. Insect cells can add compact, relatively homogenous α1–6 fucosylated Man3GlcNAc2 sugar moieties [32] of

16 kDa per glycosylation site [33]. Therefore, we suspected that the band with slower mobility in our blots corresponds to glycosylated PCFT localized at the plasma membrane, whereas the faster band corresponds to non-glycosylated PCFT. Isolation of membrane vesicles and solubilization yielded samples highly-enriched in the 43 kDa species (Fig 1B control). Consistent with glycosylation, PNGase treatment shifted the slower mobility band to a band of higher mobility (Fig 1B). In our hands, PCFT bands obtained from Sf9 whole cell lysates correspond to approximate molecular weights of 43 for glycosylated PCFT and 39 for deglycosylated PCFT.

Detergent screening for solubilization of PCFT

DDM has been widely used as a detergent to solubilize and study membrane proteins, including MFS transporters [34–43]. In pilot experiments to identify detergents suitable for solubilization of PCFT from Sf9 membranes we tested nine different detergents (concentrations in % w/v), including nonionic (0.5–2% DDM, 1–2% UDM, 0.5–2% DM, 1.25% and 4.5% OG, 1.25% NG, 1% DMNG) and zwitterionic detergents (0.5% AZ, 1% and 2.5% CHAPS and 1% FS-12). PCFT in Sf9 membranes was solubilized with these detergents, centrifuged at high-speed to separate solubilized proteins (supernatant) from unsolubilized material (pellet), separated by SDS-PAGE, and visualized by Western blot. The efficiency of solubilization was assessed by comparing the intensity of the PCFT band after solubilization to that of the control band (unsolubilized, non-centrifuged sample). PCFT in the Sf9 membranes was solubilized quantitatively (

90 to 100%) with 1 or 2% DDM, 1% FS-12 or 0.5% AZ (Fig 2). DDM consistently yielded very high efficiency in solubilization (> 70%). Based on this and its widespread use, we used DDM for all subsequent preparations.

Nine different detergents were used at the indicated concentrations to analyze solubilization of PCFT from membranes isolated from Sf9 cells. After a 2-h incubation, solubilized supernatants and pellets were analyzed using 4–15% MiniProtean TGX Precast SDS-PAGE gels, transferred to PVDF membranes and immunoblotted for detection with an antibody against the His6 tag of PCFT. First lane (Total protein) is the total amount of PCFT in Sf9 crude membranes before solubilization, and the second lane is a sample without detergent. (A) Non-solubilized pellets of the initial screen, (B) solubilized supernatants of the initial screen, and (C) solubilized supernatants with increased OG and CHAPS concentrations. See text for detergent abbreviations.

Affinity purification

We enriched PCFT from solubilized membrane proteins by liquid chromatography based on the affinity of its His6 tag for transition metals. The PCFT eluted from a TALON Co 2+ resin was significantly cleaner than that eluted from a resin containing immobilized Ni 2+ (data not shown). The His6-tagged PCFT eluted from the Co 2+ resin with 200 mM imidazole migrated at ca. 43 kDa in a 4–15% MiniProtean TGX Precast SDS-PAGE gel and showed a high degree of purity (Fig 3).

PCFT was reconstituted in liposomes as described in Experimental Procedures. Purified protein and reconstituted PCFT were subjected to SDS-PAGE (4–15% MiniProtean TGX Precast gel). (A) Protein staining (Stain-free gel, BioRad) and (B) Western blot of the same gel analyzed using an antibody against the His6 tag of PCFT. Lane 1: purified protein eluted from the Talon Co 2+ resin lane 2: PCFT reconstituted in proteoliposomes.

Size-Exclusion Chromatography (SEC) analysis

DDM-solubilized PCFT purified by immobilized Co 2+ affinity chromatography was purified further by SEC, with an overall yield of pure PCFT of

0.9 mg/l culture. Based on PCFT’s elution volume of 11.4 ml and the elution profiles of protein standards, the calculated molecular mass of the PCFT-DDM complex was

280 kDa (Fig 4) consistent with a large amount of detergent, as has been observed previously for other 12 transmembrane segment transporters [44]. Alternatively, our purified PCFT could be an oligomer, but this seems unlikely because we have shown that the monomer is the human PCFT structural/functional unit in membranes of mammalian cells and frog oocytes [26].

(A) Elution profile of PCFT solubilized in DDM. Elution volumes of standard proteins are indicated as follows: thyroglobulin (T, 669 kDa), ferritin (F, 440 kDa), aldolase (A, 158 kDa), conalbumin (C, 75 kDa), ovalbumin (O, 44 kDa). Blue dextran (BD, 2 MDa) was used for void volume determination. (B) PCFT fraction corresponding to the peak PCFT elution fraction was analyzed by protein staining (BioRad stain-free imaging). (C) The partition coefficients (Kav) of the standard proteins are plotted against the log of their molecular weights to calculate the size of the PCFT-DDM complex, yielding an apparent size of 280 kDa.

Functional characterization

Specific uptake of folic acid in Sf9 cells.

The uptake of 3 H-folic acid in Sf9 cells expressing PCFT and uninfected cells was measured over 10 min at pH 5.5 [29]. The time course of folic acid uptake was not examined in the present study, but Fig 5A shows that the uptake in cells expressing PCFT was significantly higher than in uninfected cells, and was reduced in the presence of a 200-fold excess of cold (unlabeled) folic acid (one-way ANOVA with Dunnett’s multiple comparison test, P < 0.0001). These data indicate that PCFT expressed at the plasma membrane in Sf9 cells is functional. Fig 5B shows that the uptake measured at pH 5.5 over 10 min was concentration dependent, with a Km for 3 H-folic acid uptake of 1.94 ± 0.20 mM (n = 3). This Km is similar to that reported in mammalian cells (1.7 mM in HEK 293 cells)[29] and X. laevis oocytes (1.3 mM)[9].

(A) 10-min uptake of 500 nM 3 H-folic acid (FA) by PCFT-expressing Sf9 cells determined at pH 5.5. Data are means ± SD. The value in Sf9 cells expressing PCFT (PCFT) was significantly different from that of uninfected cells (Control) (1-way ANOVA with Tukey’s multiple comparison test, P ≤ 0.0001, ****). Uptake was reduced significantly in the presence of a 200-fold excess of unlabeled folic acid (PCFT + Ex FA) (1-way ANOVA with Tukey’s multiple comparison test, P ≤ 0.0001, ****). The difference between the PCFT + Ex FA and Control was not significant (ns). (B) Concentration dependence of the 3 H-folic acid uptake in PCFT-expressing Sf9 cells (PCFT) and uninfected cells (Control). Data was fit using the Michaelis Menten equation (Graphpad Prism, San Diego, CA).

Functionality of lipid reconstituted PCFT.

Affinity purified PCFT was concentrated to 0.6 mg/ml and reconstituted in unilamellar liposomes as indicated under Experimental Procedures. The protein staining of SDS-PAGE gel and Western blot analysis in Fig 3 show the presence of PCFT in the proteoliposomes. PCFT function was demonstrated by the 30-s uptake of 300 nM 3 H-folic acid (Fig 6), where PCFT-proteoliposomes showed significantly higher uptake of 3 H-folic acid than liposomes (t-test: L vs. PCFT-PL P = 0.008, r 2 = 0.86 one preparation, n = 3). These results demonstrate reconstitution of functional PCFT in liposomes.

The 30-s uptake of 300 nM 3 H-folic acid (FA) into unilamellar PCFT-proteliposomes (PCFT-PL) was measured at pH 5.5. Unilamellar empty liposomes (L) served as control. The uptake in PCFT-PL was significantly higher than that in liposomes (t-test: L–PCFT-PL P = 0.008, r 2 = 0.86 one preparation, n = 3).


Natijalar

Peroxisome fission is important for degradation

To analyze whether peroxisome fission is important for glucose-induced selective peroxisome degradation (macropexophagy), we analyzed this process in wild-type H. polymorpha as well as in two mutant strains (dnm1 va pex11) that are strongly impaired in peroxisome fission. 19 , 20 In line with earlier observations, the levels of the peroxisomal marker protein alcohol oxidase (AO) gradually decreased in the wild-type control upon induction of macropexophagy by glucose ( Fig.ꀚ and B ). However, in both dnm1 va pex11 cells, no significant reduction of AO protein levels was observed. A similar result was obtained in the atg11 control strain, which is defective in selective pexophagy. 21 These results suggest that a reduction in peroxisome fission affects glucose-induced macropexophagy.

Figureਁ. Reduced peroxisome degradation in H. polymorpha va S. cerevisiae fission mutants. (A) Pexophagy was induced by glucose in H. polymorpha cells grown for 20 h on methanol. Equal volumes of cultures were loaded per lane. Western blots decorated with anti-alcohol oxidase (α-AO) antibodies show no significant reduction in AO levels in the peroxisomal fission mutants dnm1 va pex11, similar to the atg11 control, which is blocked in pexophagy, whereas AO levels gradually decreased in the wild-type control as expected. (B) Densitometry quantification of the blots shown in (A). The amount of AO protein present at t = 0 h was set to 100%. The bar represents the standard error of the mean (SEM). (**p < 0.01). (C) Western blot analysis showing thiolase levels of S. cerevisiae cells grown on oleate and subsequently diluted into SD(-N) medium to induce peroxisome degradation. Samples were harvested at different time points, and equal amounts of protein were loaded per lane. There is no significant decrease of thiolase in the dnm1vps1∆ double mutant where the peroxisomal fission is completely blocked. A similar result was obtained for the atg1 control strain. In the wild-type and the single vps1 va dnm1 deletion strains degradation occurred. (D) Quantification of thiolase blots shown in (C). The level of thiolase protein at t = 0 h was set to 100%. Levels were adjusted to the loading control glucose-6-phosphate dehydrogenase (not shown). The bar represents the SEM (**p < 0.01). (E) Constitutive peroxisome degradation is reduced in H. polymorpha fission mutants. Cells were grown on methanol for 16 h. Western blots were prepared using crude extracts and anti-GFP antibodies to detect Pmp47-GFP and GFP degradation products. The data show that the levels of the cleaved fusion protein (lower band) are reduced in pex11 va dnm1 cells, relative to the wild-type control. Equal concentrations of protein were loaded per lane. (F) Quantification of blots shown in (E). The levels of full-length Pmp47-GFP proteins were arbitrarily set to 100%. The levels of cleaved GFP (lower band) are indicated as percentage of the full-length fusion protein. The bar represents the SEM (*p < 0.05 **p < 0.01).

To rule out that the observed block in pexophagy is not related to the fission defect in H. polymorpha dnm1 yoki pex11 cells, but related to a direct function of fission proteins in pexophagy, we performed a control study using Saccharomyces cerevisiae. Different from H. polymorpha, in baker’s yeast, Dnm1 and Vps1 have redundant functions in peroxisome fission. Consequently, single dnm1 va vps1 mutants are only partially affected in peroxisome fission and thus can be analyzed for a direct function of these proteins in pexophagy. 22 As shown in Figureꀜ and D , single S. cerevisiae dnm1 va vps1 mutants were not blocked in glucose-induced pexophagy, whereas cells of the dnm1 vps1 double mutant, which has a major peroxisome fission defect, were impaired in peroxisome degradation.

a yordamida H. polymorpha strain that produces the peroxisomal membrane protein Pmp47 fused to green fluorescent protein (Pmp47-GFP), we analyzed constitutive peroxisome degradation in methanol-grown cells of H. polymorpha wild type and both dnm1 va pex11 mutant strains using western blot analysis and anti-GFP antibodies ( Fig.ꀞ ). As expected, in extracts of wild-type cells in addition to the band representing the full-length Pmp47-GFP fusion protein, a faster migrating band consisting of cleaved GFP was also evident, indicative of constitutive pexophagy. The ratio of the amount of cleaved GFP relative to the full-length fusion protein was reduced in dnm1 va pex11 cells compared with wild-type controls ( Fig.ꀟ ), indicating that constitutive autophagy of peroxisomes is also affected in H. polymorpha pex11 va dnm1 hujayralar. 23 Summarizing these data indicates that in yeast peroxisome fission is required for pexophagy.

Intra-peroxisomal protein aggregates affect growth and cause oxidative stress

Next, we addressed whether peroxisome fission acts in quality control of the organelles. For this we took advantage of earlier observations that production of a mutant variant of catalase, designated Cat mut , produced in wild-type H. polymorpha (i.e., also producing the endogenous catalase protein) forms enzymatically inactive protein aggregates in the peroxisomal lumen. 13 Peroxisomes containing these protein aggregates were used as a model for aberrant peroxisomes in this study.

First, we tested whether the presence of peroxisomal protein aggregates had physiological consequences. As shown in Figureꀪ cultures of the Cat mut strain showed a reduced yield. Remarkably, the specific catalase activities in cell extracts of cells of the Cat mut strain (185 U/mg) were enhanced relative to that of the wild-type control (130 U/mg). ROS measurements indicated that at all time points examined, the cells containing peroxisomal protein aggregates had enhanced ROS levels relative to the wild-type control ( Fig.ꀫ ).

Figureਂ. The effect of peroxisomal protein aggregates on growth and ROS levels. (A) Final optical densities of wild-type, dnm1 va pex11 cells, producing or not producing Cat mut , upon growth on methanol as sole carbon source for 40 h. Cell were extensively precultivated in glucose medium and subsequently shifted to medium containing methanol. Final optical densities are expressed as adsorption at 660 nm. The bar represents the SEM (*p < 0.05). (B) ROS levels in cells at different time points after the shift of glucose-grown cells to methanol medium. The mean intensity was measured by FACS. Cat mut cells show an enhanced ROS production relative to wild-type controls. The bar represents the SEM (*p < 0.05 **p < 0.01).

Intraperoxisomal protein aggregates are removed by fission and degradation

To substantiate whether the protein aggregates induce peroxisome fission, we introduced Cat mut in the H. polymorpha atg1 mutant, in which autophagic degradation of peroxisomes is blocked. 24 In order to visualize peroxisomes we introduced the fluorescent peroxisomal membrane marker Pmp47-GFP. Fluorescence microscopy analyses of cells, cultivated for 16 h on methanol, revealed that the Cat mut -producing atg1 strain contained, in addition to the normal organelles, relatively small organelles that were not observed in the atg1 parental strain ( Fig.ꀺ ). This result was confirmed by electron microscopy analysis of KMnO4-fixed cells ( Fig.ꀻ ) and also showed the presence of aggregates in the small organelles ( Fig.ꀻ ). Subsequent analysis, however, revealed that small organelles were not evident by fluorescence microscopy of wild-type cells producing Cat mut ( Fig.ꀺ ), whereas electron microscopy revealed that these cells harbored fewer small aggregate-containing peroxisomes relative to the atg1 strain producing Cat mut . Apparently, the small separated organelles were rapidly removed in the wild-type background. This process was further analyzed by electron microscopy. These analyses revealed that once formed, aggregates migrated to the periphery of the organelle where they were included in the buds that subsequently split off from the mother organelle ( Fig.ꀼ ).

Figureਃ. Microscopy analysis of H. polymorpha atg1, atg1-Cat mut and wild-type-Cat mut cells. Cells were grown on glycerol/methanol for 16 h. Peroxisomal membranes are marked by Pmp47-GFP. (A) Relative to the atg1 control, the atg1-Cat mut strain contains multiple small peroxisomes, which was not evident in wild-type-Cat mut cells. The bar represents 1 µm. The peroxisomal phenotype was confirmed by electron microscopy (B). Note the presence of protein aggregates in many of the organelles in atg1-Cat mut cells of which fewer are observed in wild-type-Cat mut cells. The bar represents 0.5 µm. (C) Ultrathin sections of KMnO4-fixed atg1-Cat mut cells showing different stages of the formation of a small peroxisome containing a protein aggregate. The bar represents 0.2 µm. (D) Electron micrographs, showing details of dnm1.atg1 cells (left panel) and pex11.atg1 cells (right panel) producing Cat mut , demonstrating the presence of aggregates in the enlarged peroxisomes in these cells. The bar represents 0.2 µm. P, peroxisomes M, mitochondria N, nucleus V, vacuole.

To further address their degradation, we cultivated the H. polymorpha Cat mut strain on a mixture of glycerol/methanol and subsequently administered 1 mM of the protease inhibitor phenylmethanesulfonylfluoride (PMSF) to the culture to reduce the rate of degradation of autophagic bodies in the vacuole. Electron microscopy analysis of these cells showed that at the onset of the experiment vacuoles in Cat mut -producing cells contained very low numbers of autophagic bodies ( Fig.ꁊ and B ). However, after 2 ( Fig.ꁌ and D ) and 4 h ( Fig.ꁎ and F ) of PMSF administration the accumulation of aggregate containing organelles in the vacuole was evident. These structures were not observed in the wild-type control. The presence of catalase protein was confirmed by immunocytochemistry ( Fig.਄G and H ). These data suggest that peroxisomes with protein aggregates are delivered to the vacuole for autophagic degradation. In conclusion, we showed that protein aggregates, which accumulated in the peroxisomal lumen are removed by concerted fission and degradation events.

Figure਄. Catalase aggregates are degraded by autophagy. H. polymorpha wild-type and Cat mut cells were cultivated on glycerol/methanol for 12 h and subsequently treated with 1 mM PMSF (t = 0). Electron micrographs of KMnO4-fixed Cat mut cells [at t = 0 h (B)], [at t = 2 h (D) and t = 4 h (F)] revealed progressive accumulation of autophagic bodies containing peroxisomes with protein aggregates that are not observed in wild-type cells (A, C and E). Immunocytochemical localization of catalase shows that labeling is confined to peroxisomes of wild-type cells (G) and is also abundant in the vacuole of Cat mut cells (H). M, mitochondria N, nucleus P, peroxisomes V, vacuole. The bar represents 0.5 µm.

Degradation of aggregates is reduced in dnm1, pex11 and atg11 hujayralar

Clearly, the separation of small aggregate-containing peroxisomes is different from the fission events that participate in normal peroxisome proliferation in wild-type cells. Therefore, we analyzed if this fission process depends on Dnm1 and Pex11. 19 , 20 To this end, corresponding deletion stains were constructed that produced Cat mut N-terminally fused to GFP (GFP-Cat mut ) to fluorescently mark the protein aggregates. Fluorescence microscopy analysis of cells also producing the DsRed-SKL peroxisomal matrix marker protein revealed that small green fluorescent spots were present in peroxisomes of both mutant strains, as well as in the wild-type control ( Fig.ਅ ). To analyze the possible presence of GFP-Cat mut in the vacuole, the red vacuolar marker FM 4� was used instead of DsRed-SKL ( Fig.ਆ ). Fluorescence microscopy analysis confirmed that green fluorescence was observed only infrequently in vacuoles of dnm1 va pex11 cells relative to the wild-type control ( Fig.ਆ ). Western blot analysis using crude extracts of these cells confirmed reduced cleavage of GFP-Cat mut relative to that in the wild-type cells producing GFP-Cat mut ( Fig.ਇ ). The reduced degradation of the aggregates also affected growth of the strains as lower growth yields on methanol were observed in both dnm1 va pex11 cells for the strains producing Cat mut ( Fig.ꀪ ).

Figureਅ. Visualization of catalase protein aggregates. H. polymorpha wild-type, dnm1 va pex11 cells, producing DsRed-SKL and GFP-Cat mut , were grown on methanol for 16 h. The peroxisomes contained GFP spots in the peroxisomal lumen, which represent the protein aggregates.

Figureਆ. Visualization of catalase protein aggregates. Identical experiment as shown in Figureਅ , using wild-type, dnm1 va pex11 cells, producing GFP-Cat mut stained with the vacuolar marker dye FM 4�. GFP fluorescence was frequently observed in the vacuoles of wild-type cells. GFP fluorescence was also observed in the vacuoles of the dnm1 va pex11 cells, albeit at reduced numbers compared with the wild-type control. The bar represents 1 µm.

Figureਇ. Autophagic degradation of GFP-Cat mut is reduced in pex11 va dnm1 hujayralar. (A) Western blot analysis using crude extracts of cells described in Figureਅ , decorated with anti-GFP antibodies. All strains contain both the full-length GFP-Cat mut protein together with GFP (arrow), due to cleavage of the fusion protein. Pyruvate carboxylase (Pyc1) was used as a loading control. (B) Quantification of the levels of GFP-Cat mut and GFP protein of the blots shown in (A). The level of full-length GFP-Cat mut is set to 100%. The bar represents the SEM (**p < 0.01). (C) Quantification of GFP fluorescence in the vacuole. The percentage of cells containing GFP fluorescence in the vacuole was calculated for wild-type, dnm1 va pex11 cells containing PAOXGFP-Cat mut . Per strain 2 samples of each 100 cells were counted The bar represents the SEM (*p < 0.05).

To strengthen the observation that Dnm1 and Pex11 are indeed required for the separation of the small aggregate-containing peroxisomes, we also performed electron microscopy analysis of dnm1 atg1 va pex11 atg1 double-mutant cells producing Cat mut . As evident from Figureꀽ , these cells harbor large peroxisomes that contain protein aggregates.

Degradation of the aggregates was also reduced upon deletion of ATG11, a gene involved in selective peroxisome degradation. In atg11 cells producing GFP-Cat mut numerous green spots were observed ( Fig.ꂊ ). However, vacuoles did not accumulate GFP as observed in the wild-type background (compare Fig.ਆ and Fig.ꂋ ). The block in autophagic degradation was furthermore evident from western blot analysis of these cells showing that cleavage of GFP from the GFP-Cat mut fusion protein was strongly reduced ( Fig.ꂌ ).

Figureਈ. Autophagic degradation of GFP-Cat mut is reduced in atg11 hujayralar. (A) ichida H. polymorpha atg11 cells producing DsRed-SKL and GFP-Cat mut and grown on methanol for 16 h, most GFP-Cat mut spots colocalize with the peroxisomal matrix marker DsRed-SKL. Some of the green spots do not colocalize with DsRed-SKL. Most likely this is the result of the asymmetric peroxisome fission process, resulting in the formation of small aggregate-containing peroxisomes that contain no or very little DsRed-SKL. (B) Vacuolar staining of atg11 GFP-Cat mut cells with FM 4� dye. GFP fluorescence in the vacuoles is reduced in atg11 cells relative to the wild-type control (see Fig.ਆ ). The bar represents 1 µm. (C) Western blots decorated with anti-GFP antibodies fail to demonstrate the GFP cleavage product (arrow) in atg11 cells producing GFP-Cat mut .


NegativeArraySizeException

This error usually means that your image planes are larger than the maximum supported size.

The original ImageJ only supports image planes with 2 gigapixels (2^31 = 2147483648 pixels in case of a square image, the maximum allowed is 46340 x 46340 pixels) or less. If your data has extremely large image planes—e.g., 50000 x 50000 pixels—you may need to analyze region by region. One way to do this is using the “Crop on import” feature of the Bio-Formats plugin.

If you are using Bio-Formats to open a file, however, the size limit is a bit more complicated. Instead of using short[] as in ImageJ, Bio-Formats store data in byte[] when reading planes. If the source image is in 16 bit or in 32 bit (4 bytes, eg. floating point TIFF), the maximum pixel numbers allowed per plane will be 1/2 (1 gigapixels) or 1/4 (0.5 gigapixels), respectively.

ImageJ2 supports larger image planes internally, but uses the original ImageJ user interface by default, which once again limits visualization to 2 gigapixels. The ImageJ2 team is working to lift these size restrictions see imagej/imagej#87.


Usullari

Plant materials and growth conditions

Arabidopsis (Columbia-0) and the T-DNA insertion mutant line (SALK_056011, locus At4g17740) were obtained from the Arabidopsis Resource Center (Columbus, OH). Seedlings were grown on 1/2 MS medium containing 3.0% sucrose (pH 5.7) for 4 weeks under the conditions of 16 h light / 8 h dark cycle and 20 μmol photons m − 2 s − 1 light intensity during the light periods.

Blue native PAGE and 2D SDS-PAGE

Chloroplasts were extracted from 50 wild-type and 50 atctpa-mutant plants. Blue native gel electrophoresis was performed as described previously [25, 38]. For 2D SDS-PAGE, the blue native gel lanes were excised with a razor blade and incubated in 2 × SDS sample buffer containing 2.5% (vol / vol) β-mercaptoethanol (β-ME) for 20 min at 75 °C, then for 20 min at 25 °C. Lanes with denatured proteins were placed on top of 12% SDS gels, then subjected to the second dimensional separation.

Immunoblot analysis

For immunoblotting, protein samples were separated on 12% SDS gels and transferred to nitrocellulose membranes (BioTrace™ NT nitrocellulose, Mexico) followed by a western blot analysis. After blocking with 5% milk, the membranes were subsequently incubated with primary antibodies generated against the indicated proteins and detected using the Super Signal™ West Pico PLUS Chemiluminescent Substrate kit (Thermo Scientific, USA).

Yeast two-hybrid assay and growth curves analysis

The yeast two-hybrid assay was performed using the Split Ubiquitin System (DUAL membrane, Dualsystems Biotech) as described previously [39, 40]. The mature D1 (amino acids 1–344) and pD1 (amino acids 1–353) were cloned into pCCW-STE vector (encoding the Cub-LexA-VP16 fragment) as the bait for interaction assay. CP43, CP47 and D2 were cloned into pDSL-Nx vector (encoding the NubG fragment) as the prey for the assay. Yeast strain NMY32 was co-transformed with the bait and prey constructs, respectively. The interactions were determined by the growth of yeast cells on agar plates with Synthetic Defined (SD) medium lacking Trp, Leu, His, and adenine (SD-Trp Leu His Ade, FunGenome) without or with 2 mM 3-amino-1, 2, 4-Triazole (3-AT). The growth curves of liquid cultured yeast cells were obtained by measuring the absorbance at 600 nM (OD600). Six colonies of each transformation were cultured in SD-Trp Leu His Ade medium containing 30 μg / ml kanamycine. OD600 values were recorded at various time points. pDSL-Nx vector was used as the negative control, and NubI was used as the positive control.

Statistik tahlil

ImageJ (https://imagej.nih.gov/ij/) was employed to qualify the distribution ratio of PSII subunits among different subcomplexes.


Videoni tomosha qiling: western blot analysis with imageJ (Iyul 2022).


Izohlar:

  1. Chadburn

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