Molbiol.ru | О проекте | Справочник | Методы | Растворы | Расчёты | Литература | Орг.вопросы Web | Фирмы | Coffee break | Картинки | Работы и услуги | Биржа труда | Zbio-wiki NG SEQUENCING · ЖИЗНЬ РАСТЕНИЙ · БИОХИМИЯ · ГОРОДСКИЕ КОМАРЫ · А.А.ЛЮБИЩЕВ · ЗООМУЗЕЙ Темы за 24 часа [ Вход* | Регистрация* ] Форум: | |
Evgenij Evdokimov Springfield, VA |
Bydy ochen' priznatelen esli podskazhete kak mozhno reshit' problemy: na commassie blue belki nikak ne pojavljajutsja, tol'ko western. projavka strepavidin conjugated alkaline phosphatase. Mozhno li izvlech' belki iz membrani (nitrocellulose ili PVDF) i potom sdelat' na nih MS??? Strepavidin-AP meshat' tozhe bydet, navernoe... [Текст переведён с транслита] |
Husker Постоянный участник |
а кроме того для MS , белок должен быть в геле, а не на мембране [ 11-10-2002: Сообщение отредактировано: Husker ] |
Andrei Постоянный участник |
Ещё можно окрашивать белки в геле флюоресуирующими красителями-посмотрите в каталоге фирмы Molecular Probes, www.probes.com Чувствительность у них выше, чем у Кумасси, и фирма утверждает, что приближается к серебру. Какие-то из них подходят для MS. По поводу извлечения белков из мембраны...что-то подобное делали в ИБХ лет 12 назад, методики я к сожалению не помню. Кажется, нитроцеллюлозу растворяли в ацетонитриле, потом белок сорбировали на обращённой фазе и элюировали.# |
Husker Постоянный участник |
|
Lex Постоянный участник |
A) If you wanna sequence your whole peptide - use the following: * Transfer gel-sparated proteins onto PVDF membrane (no detergent in transfer buffer)) and place in centrifuge tubes. * Wash with 50% CAN containing 0.1%TFA (2 times, 100ul each) * Add 85ul of 80%acetic acid * Add 15 ul BNPS(1 mg in 250 ul acetic acid) solution. * Incubate for 2 to 3 hours at 65oC or 1 hour at 80oC in the dark. * Remove the reaction solution, and wash the PVDF membrane with 80%acetic acid, twice, 100 ul each. * Wash with 10% methanol, once 1 ml. * Load onto protein sequencer (note: sample is loaded wet). What I advise you to follow is to digest your protein IN pvdf, then isolate it and proceed to sequencing. B) The protocol is the following: 1) probe your membrain with any appr. ab, figure your band shape, pierce it by pin 2) strip your membrane in mild conditions Wet membrane with 100%MeOH 3)Soak membrane in 10 ml Reducing Solution at 50C for 10 minutes 4) Place membrane in 10 ml Alkylating Solution at 50C for 5 minutes 5)Rinse membrane 1 X with 25 ml 100 mM Tris pH 9.2 6)Rinse membrane in 15 ml Digestion buffer 7) Cut the pinned area of your PVDF membrane into 1 x 1 mm squares (use a fresh razor blade). Attn.! put gloves on! My collaborator always complains that t90% of artifacts come from keratin stuff. 8) Incubate in 20-2000 ul (depends on your initial prep) Digestion buffer; mix; 30 min RT (important to use smallest volume possible yet still cover membrane) 9) Add 0.2 mgm protease (0.1 mgm/ml). Incubate 40C for 18-24hours. Spin sample briefly to collect liquid; sonicate 5 min at 30°C 10)Remove super with tip to siliconized test tube 11)Add 500 ul of digestion buffer, vortex and sonicate 5 min at 30°C 12)Remove supernatant fluids and pool with first supernatant materials 13) repeat steps 7-8 two times 14) Add 100 ml of 0.1% TFA to the membranes; vortex and sonicate 5 min at 30°C 15)Remove supernatant fluids and pools with first supernatant materials 16) speedvac it up to a small (40ul) volume your protein fragments are ready for MS P.S. I was able to get sequenced 5 pmol mid-size proteins P.P.S. Digestion Buffers (all these are common, I just forgot where I got the above protocol) 50 mM Tris pH 9.2, 1% Reduced Triton X-100, 10% Acetonitrile C) 100 mM ammonium bicarbonate, 1% Reduced Triton X-100,10% Acetonitrile (for trypsin) Reducing Solution: Add 10 ml (1M stock) DTT to 0.99 ml PCS2 Alkylating Solution: Add 1.42 gm acrylamide to 8.2 ml PCS2 PCS2: Guanidine-HCL, pH 8.3 [ 11-10-2002: Сообщение отредактировано: Lex ] [ 11-10-2002: Сообщение отредактировано: Lex ] |
123goal 123goal Постоянный участник |
|
123goal 123goal Постоянный участник |
|
123goal 123goal Постоянный участник |
|
123goal 123goal Постоянный участник |
|
123goal 123goal Постоянный участник |
|
123goal 123goal Постоянный участник |
|
jaipurescorts0010 Постоянный участник |
Known as the "Lake City of India," Udaipur has a rich cultural history. Udaipur attracts visitors from throughout the world who want to see its undiscovered splendours. Also, some people travel to Udaipur to escape the stress and commotion of daily life. Therefore, some individuals find that travelling to new places is a great way to unwind, while others find that interacting with new people is a fun way to socialise. You've found the ideal website if you're enthusiastic and seeking for We have a group of knowledgeable and experienced call girls in Udaipur that are experts in their business and have years of expertise. Our call ladies are here to satisfy all of your wishes and provide you the most satisfaction if you're in Udaipur and feeling horny. Our youthful, attractive, and seductive Prepare yourself for an unforgettable encounter when you use the Udaipur Escort Service. People are drawn to these young, stunning call girls because of their appearance. But what about those who are unfamiliar with the process for reserving a call lady in Udaipur? We are here for them to help them. If such is the case, there is no need to be concerned since you may hire the ideal female from our a nudist service. You will most surely feel sexually attracted by our Udaipur escorts after meeting them. Your dick may become sensitive and erect, and you could experience wetness around your penis. As a result, if our One of the things that many males like doing is hooking up with young, attractive call ladies. Also, some people like spending time with call ladies. Because of this, our call girls in Udaipur are accessible day and night and make a great companion. You may get in touch with our dependable and genuine Udaipur Escorts right away if you want to meet with cute and youthful |
123goal 123goal Постоянный участник |
|
123goal 123goal Постоянный участник |
|
123goal 123goal Постоянный участник |
|
123goal 123goal Постоянный участник |
|
123goal 123goal Постоянный участник |
|
123goal 123goal Постоянный участник |
|
123goal 123goal Постоянный участник |
|
Udaipur Escorts IP-штамп: frQrtJ1xVpdzY гость |
Welcome to UDAIPUR Escorts , the home of the best Russian female escorts offering 24-hour incall and outcall services for your convenience. Our extensive Russian escort, webcam and phone sex directory features sexy and insatiable female companions that are highly skilled, talented and experienced in adult work. Whatever your secret fantasy may be, our delectable UDAIPUR Escort deliver the ultimate service to ensure it becomes a reality. If You are Intersted of Escort Girls in Udaipur Then Book This Website . |
123goal 123goal Постоянный участник |
|
carbide insert quotation IP-штамп: fr4iSIt6irJd6 гость |
|
123goal 123goal Постоянный участник |
|
123goal 123goal Постоянный участник |
|
123goal 123goal Постоянный участник |
|
123goal 123goal Постоянный участник |
|
123goal 123goal Постоянный участник |
|
123goal 123goal Постоянный участник |
|
123goal 123goal Постоянный участник |
|
123goal 123goal Постоянный участник |
|
123goal 123goal Постоянный участник |
|
123goal 123goal Постоянный участник |
|
123goal 123goal Постоянный участник |
|
123goal 123goal Постоянный участник |
|
123goal 123goal Постоянный участник |
|
123goal 123goal Постоянный участник |
|
123goal 123goal Постоянный участник |
|
123goal 123goal Постоянный участник |
|
123goal 123goal Постоянный участник |
|
123goal 123goal Постоянный участник |
|
123goal 123goal Постоянный участник |
|
123goal 123goal Постоянный участник |
|
123goal 123goal Постоянный участник |
|
123goal 123goal Постоянный участник |
|
123goal 123goal Постоянный участник |
|
123goal 123goal Постоянный участник |
|
123goal 123goal Постоянный участник |
|
123goal 123goal Постоянный участник |
|
123goal 123goal Постоянный участник |
|
123goal 123goal Постоянный участник |
|
« Предыдущая тема · Молекулярная и клеточная биология · Следующая тема » |