antibody stripping buffer

Western Blot Stripping Buffer

Szczegółowe informacje o produkcie
Wykonywanie elektroforezy żelowej i duplikowanie testów immunoblot w celu sprawdzenia nowych głównych przeciwciał lub stężeń przeciwciał jest czasochłonne i kosztowne. Restore Western Blot Stripping Buffer eliminuje to marnotrawstwo podczas wykrywania immunoblotów za pomocą chemiluminescencyjnych substratów Western blotting. Restore Stripping Buffer zapewnia przejrzystą i przyjazną dla środowiska eliminację głównych i drugorzędowych przeciwciał z immunoblotów bez eradykacji lub uszkodzenia unieruchomionego antygenu, co pozwala na swobodne usuwanie i ponowne sondowanie blotów.

Wykrywanie chemiluminescencji metodą Western blot z użyciem odczynników równoważnych z SuperSignal Substrates firmy Thermo Scientific dla peroksydazy chrzanowej jest prawdopodobnie jedną z najczęstszych i najdelikatniejszych strategii stosowanych natychmiast. W wyniku tego, że te substraty nie wytrącają się i nie wiążą się z powierzchniami błon, Western blot wykrywane przez chemiluminescencję można usunąć za pomocą odczynników, które usuwają przeciwciała główne i drugorzędowe związane z powinowactwem. Aby był skuteczny, bufor strippingowy musi być wystarczająco mocny, aby oddzielić pewne przeciwciała, ale wystarczająco lekki, aby oddzielić przeniesione białka docelowe w stanie nienaruszonym na błonie nitrocelulozowej lub PVDF. Przywróć Western Blot Stripping Buffer ma te cechy.

Przez usuwanie i ponowne sondowanie nie ma czegoś takiego jak marnowanie nietypowych lub drogich próbek na pracę z wieloma żelami z zamiarem sondowania różnych celów. Pojedyncza membrana z jednego żelu może zostać usunięta za pomocą buforu Restore Western Blot Stripping Buffer, aby usunąć pierwsze przeciwciała. Rozbiór blotu zajmuje tylko 15 do 30 minut, w oparciu o powinowactwo pierwszego przeciwciała. Po usunięciu, zablokuj i ponownie sonduj za pomocą zupełnie nowego głównego przeciwciała. Alternatywnie, odcisk można usunąć i ponownie sondować z dostosowanymi stężeniami przeciwciał, aby zoptymalizować okoliczności po uzyskaniu początkowo słabych wyników.

 

The Western BLoT Stripping Buffer is an answer for eradicating main and secondary antibodies from probed Western blot membranes. Antibody elimination with this buffer can happen underneath gentle circumstances (room temperature, 30 min incubation), minimizing lack of immobilized protein from the membrane. When utilizing a PVDF membrane, the identical membrane might be stripped and reprobed 2–5 occasions. After stripping, membranes might be re-probed, both with a unique focus of main antibody or with a wholly totally different main antibody.

Stripping to okres czasu używany do wyjaśnienia eliminacji głównych i drugorzędowych przeciwciał z błony Western blot. Stripping jest korzystny, gdy trzeba zbadać wiele białek na identycznym blocie, jako ilustrację białka ciekawości i zarządzania ładowaniem. Podczas sondowania wielu celów, usuwanie i ponowne sondowanie pojedynczej membrany jako substytut pracy i blottingu wielu żeli ma tę zaletę, że oszczędza próbki, zapasy i czas.

Nie jest wskazane dokonywanie ilościowych porównań celów sondowanych przed i po strippingu, ponieważ proces ten usuwa z błony pewne białko wzorcowe. Z podobnej przyczyny nie należy badać pozbawionej błony membrany pod kątem braku białka.

Proces

  1. Podgrzej bufor do 50°C
  2. Dodaj bufor do małego plastikowego pola, które ma przyzwoitą pokrywkę; użyj ilości, która może osłaniać membranę
  3. Dodaj membranę. Inkubować w temperaturze 50°C nawet przez 45 minut z lekkim mieszaniem
  4. Wyeliminuj odpowiedź zgodnie z wymaganiami dla buforów opartych głównie na ß-merkaptoetanolu
  5. Opłucz membranę pod pracującym kranem z wodą przez 1–2 min
  6. Ślady ß-merkaptoetanolu uszkadzają przeciwciała. Pierz intensywnie przez pięć minut w TBST
  7. Przygotowany do blokowania
antibody stripping buffer
antibody stripping buffer

Thermo Scientific Restore Western Blot Stripping Buffer bezpiecznie i skutecznie usuwa główne i drugorzędowe przeciwciała z membran nitrocelulozowych i PVDF, umożliwiając ponowne badanie chemiluminescencyjnych Western blot.

Opcje przywracania buforu Western Blot Stripping Buffer:

• Oszczędza czas — nie trzeba ponownie uruchamiać żeli i blotów
• Oszczędza kosztowny wzór — ponownie sonduje membranę przy użyciu identycznego wzorca docelowego
• Wydajny — preparat jest bardziej przyjazny dla środowiska przy usuwaniu przeciwciał niż samodzielnie wykonane bufory
• Lekkie — nie uszkadza docelowego antygenu podczas usuwania, co umożliwia przyjazne dla środowiska ponowne obsadzanie
• Bez zapachu — brak merkaptanów oznacza brak gryzących zapachów
• Ekonomiczny — tańszy niż inne przemysłowe bufory do usuwania


Funkcje
• Ponowne użycie membrany nitrocelulozowej lub PVDF w celu wykrycia unikalnego celu z unikalnym głównym przeciwciałem
• Ponowne sondowanie blotu w celu odpowiedniego lub zoptymalizowania stężeń przeciwciał, które były nieskuteczne w czasie podstawowym

Streszczenie protokołu
• Wash blot w celu usunięcia substratu chemiluminescencyjnego.
• Inkubować odcisk w buforze Restore Western Blot Stripping Buffer przez pięć do 15 minut w temperaturze 37°C (w przypadku niektórych przeciwciał wystarcza temperatura pokojowa).
• Wyjmij blotting i umyj w buforze do płukania (TBS-T lub PBS-T).
• Przyjrzyj się wystarczającej eliminacji przeciwciał.
• Przeprowadź kolejny eksperyment immunoblot.

 

 

 

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