* работать только в перчатках;
* качество воды очень критично, на всех этапах использовать только mQ.
нельзя использовать b-MeEtOH в качестве агента, восстанавливающего дисульфидные связи: он вызывает появление двух артефактных полос в районе 50 и 67kDa.
сообщалось о возможности повысить чувствительность метода в 2-9/40-50 раз с помощью двойного окрашивания: предварительное окрашивание Coomassie Blue (нормальная/колоидная форма), а затем - серебряное окрашивание.
Вещества, влияющие на окрашивание:
SDS
Глицин
Tris
Мочевина
Глицерин
Уксусная кислота
Белки - вызывают сильное фоновое окрашивание
По пунктам
п. 6.
К NH4(35%) добавить 0.1M NaOH, затем, перемешивая, понемногу добавлять AgNO3. Когда выпадет коричневый осадок, растворить добавлением минимального количества NH4. Довести объем до 100 ml H2O. Раствор нестабилен, должен быть использован в течение 5'. окрашивание проводится только в перчатках.
п. 8.
После 10' существенно возрастает фон.
Проявление продолжается ещё 2-4' после удаления геля из проявителя.
A01. According to Oakley BR et al. (1980) Anal. Biochem., 105, 361-363. Optimized for PAAG slabs 140 X 140 X 0.8 mm. Step 01. Wash gel with 50 % ethanol, 10 % acetic acid for 45 min Step 02. Wash gel with 5 % methanol, 5 % acetic acid for 45 min Step 03. Wash gel with 10 % glutaraldehyde solution in water for 30 min Step 04. Wash gel with 0.5 - 1 l water, 3 times X 10 min Step 05. Leave gel in water for 2 - 12 h Step 06. Prepare 100 ml stain solution: under permanent stirring add 1.4 ml concentrated water solution of NH3 to 21 ml 0.36 % NaOH, then slowly add 4 ml 19.4 % AgNO3 (20 g AgNO3 + 100 ml H2O). Brown precipitate can be seen, but it should disappear quickly. Adjust volume to 100 ml with water Step 07. Add 100 ml freshly made stain solution to the gel, shake gel gently 15 min Step 08. Rinse gel once with 0.5 - 1 l water for 2 min Step 09. Develop gel with freshly made 0.005 % solution of citric acid in 0.019 % formaldehyde, 10 - 15 % methanol. Step 10. Wash gel with 0.5 - 1 l water, 3 times X 20 min Comment 1. Sensitivity is about 0.1 ng of protein per band. There is relatively high background staining. Comment 2. After staining gel becomes fragile. Prior drying: wash gel with 30 % sodium thiosulfate for 15 min, then put into methanol : water : glycerol = 70 : 27 : 3 (v/v/v) for 5 min.
A02. According to Merril et al. (1981) Science, 211, 1437-1438. Photochemical staining. Optimized for 0.8 mm -thick PAAG slabs. Step 01. Fixation. Put gel in 50 % methanol, 12 % acetic acid for 20 min Step 02. Wash gel with 10 % ethanol, 5 % acetic acid, 3 times X 10 min Step 03. Wash gel with 3.4 mM potassium dichromate (K2Cr2O7), 3.2 mM HNO3 Step 04. Rinse gel quickly with water, 4 times X 30 sec Step 05. Put gel in 12 mM AgNO3 for 30 min, and during first 5 min illuminate gel intensively by a closely placed fluorescent lamp Step 06. Development. Wash gel 2 - 3 times with 0.28 M Na2CO3, 0.5 ml/l 37 % formaldehyde, until appearance of the bands Step 07. Stop gel development with 1 % acetic acid Step 08. Rinse gel with water, then wash with 3 % glycerol and dry Comment 1. Attempt to optimise silver staining according to Speed/Sensitivity criterion. Sensitivity is 0.02 - 0.05 ng/mm2 of protein (detection limit is about 0.01 ng/mm2 of protein). Comment 2. Increase of sensitivity due to illumination takes place only when the gel is less than 1 mm thick. Comment 3. Merril mentions in another article that the mild "amber" light provides better sensitivity.
A03. According to Merril et al. (1981) Anal. Biochem., 110, 201-207. Photochemical staining. Step 01. Fixation. Put gel in 50 % methanol, 12 % acetic acid for 10 min Step 02. Wash gel with 10 % ethanol, 5 % acetic acid, 2 times X 5 min Step 03. Wash gel with 0.5 % Potassium Ferricyanide (K3[Fe(CN)6]) for 5 min Step 04. Rinse gel quickly with water, 3 times Step 05. Put gel in freshly made stain solution: 2 g/l AgNO3, 2 g/l NH4NO3, 5 ml/l 37 % formaldehyde, 0.6 mg/l benzotriazole (to prevent fogging), illuminate gel in solution with a 1500 Watt tungsten lamp for 20 min Step 06. Development. Put gel in 3 % Na2CO3, 1 ml/l 37 % formaldehyde, 1.2 mg/l benzotriazole. Continue illumination. Bands appear in approx. 1 min. Change developing solution 2-3 times until desirable band intensities will be achieved. Step 07. Stop gel development with 1 % acetic acid, then wash gel with water. Comment 1. The procedure takes only 1 h, sensitivity is as high as in case of A01 method. Comment 2. After step 05 there is no need to rinse gel with water.
A04. According to Merril et al. (1984) Electrophoresis, 5, 289-297 Photochemical staining. Step 01. Fixation. Put gel in 50 % methanol, 10 % acetic acid, 20 g/l citric acid, 2 g/l NaCl for 5 min Step 02. Wash gel with water few times Step 03. Put gel in 20 g/l AgNO3, 50 % methanol, 10 % acetic acid, illuminate gel in solution with 160 Watt fluorescent lamp or 500 Watt tungsten lamp until band appearance Step 04. Wash gel with water Comment 1. Procedure is simple and takes only 10 - 15 min. Comment 2. Method requires sufficient amount of AgNO3. Comment 3. Sensitivity is 0.5 ng of protein per band. Comment 4. Development is completely illumination-dependent, therefore it stops when the lamp is turned off. There is no need to treat the gel with any special stop solution.
A05. According to Sammons et al. (1981) Electrophoresis, 2, 135-141. Colour staining after protein gel-electrophoresis (both native and SDS PAGE). Optimized for 1.5 mm -thick PAAG slabs. Step 01. Fixation. Put gel in 50 % ethanol, 10 % acetic acid for 2 - 12 h. Long wash is necessary primarily for the complete SDS removal Step 02. Wash with a fresh portion of 50 % ethanol, 10 % acetic acid for 2 h Step 03. Wash with 25 % ethanol, 10 % acetic acid, 2 times X 1 h Step 04. Wash with 10 % ethanol, 0.5 % acetic acid, 2 times X 1 h. Gel can be left overnight in the last solution Step 05. Wash with 1.9 g/l AgNO3 for at least 2 h Step 06. Rinse quickly with water (10 - 20 sec) Step 07. Develop gel with freshly prepared solution: 87.5 mg/l NaBH4, 7.5 ml/l 37 % formaldehyde, 0.75 M NaOH. Shake gel gently for about 10 min Step 08. Colour enhancement. Wash gel with 7.5 g/l Na2CO3 (minimum 2 times X 1 h). Maximum of the colour intensity is to be achieved after 6 h of Na2CO3 diffusion into the gel. Comment 1. Optimal ratio of any washing solution volume to the gel volume is 11 : 2 (in case of AgNO3 solution - 3 : 1). Comment 2. Steps 05 - 08: all solutions should be deaerated prior use. Comment 3. Polypeptide bands can be coloured in blue, green, yellow and red. Colour depends on polypeptide composition: for example, bands of basic low-molecular-weight peptides are usually green. Comment 4. When a band contains too much protein (above 500 ng/band), colour can be seen only on the edge of the protein spot, the central part of the band is "empty", so that the band after staining look like an oval ring.
A06. According to Heukeshoven, Dernick (1985) Electrophoresis, 6, 103-112. Method is optimised for protein staining in ultrathin (UTL, 100-300 micrometers) SDS-PAAG slabs (UTLSDS) and IEF slabs (UTLIEF), but can be used for conventional SDS and IEF gels. Step 01. Fixation. UTLSDS - 30 % ethanol, 10 % acetic acid, at least 30 min. Conv. SDS - 30 % ethanol, 10 % acetic acid, at least 3 h. UTLIEF - 20 % trichloroacetic acid, for 10 - 20 min, then 2.5 % glutaraldehyde, 1 M Sodium Acetate, for 30 min. Conv. IEF - 20 % trichloroacetic acid, for 30 - 60 min Step 02. Wash 2 times with 10 % ethanol, for 5 min (UTL) or 10 min (conv.) each time Step 03. Wash 3 times with water, for 5 min (UTL) or 10 min (conv.) each time Step 04. Wash with 0.5 % AgNO3 (UTL) or 0.1 % AgNO3 (conv.) for 30 min Step 05. Rinse with water for 10 - 20 sec Step 06. Develop gel with 2.5 - 3 % Na2CO3, 0.02 % formaldehyde for 5 - 10 min Step 07. Stop gel development by washing with 1 % acetic acid for 5 min. Step 08. Wash with water, 3 times X 10 min (UTL - 5 min) Step 09. Rinse with 0.5 % Farmer's reducer for 10 - 30 sec. Dry Farmer's reducer consists of 30 weight % of Ferricyanide (K3[Fe(CN)6]), 60 weight % of Sodium Thiosulfate and 10 weight % of Na2CO3 Step 10. Wash with water flow for 1 min Step 11. Wash with water, 3 times X 10 min (UTL - 5 min) Step 12. Repeat cycle from Step 04, if necessary. Comment 1. The use of ethanol instead of methanol upon fixation (step 01) improves quality of staining. Comment 2. SDS should be completely removed from the gel prior AgNO3 treatment. Therefore it is recommended to leave gel in the fixation solution overnight. Comment 3. The use of glutaraldehyde (Step 01) decreases protein losses in case of ultrathin gels. Comment 4. No light stimulation of staining process has been observed (unlike A02 and A03). Comment 5. Steps 09 - 12 are optional. Relatively mild reduction with diluted Farmer's reducer results in destaining of the gel surface and significantly improves contrast. Farmer's reagent is subproportional reducer, i.e. it destains background stronger than the bands. There is always a possibility to repeat staining after complete silver reduction. Reducer must be removed prior AgNO3 treatment, otherwise background staining will be high. Comment 6. Sensitivity - in case of 1 mm-thick SDS PAAG: 0.5 - 1 ng of protein per band, in case of UTLIEF: 30 - 70 ng of protein per band. Comment 7. Silver staining can be performed after preliminary Coomassie staining, but in this case Coomassie dye must be washed out with a few portions of 30 % ethanol, otherwise background will be too high. After washing procedure should be started from Step 02. Comment 8. After staining gel should be washed with water and dried. Comment 9. Contribution of different amino acyl residues to the intensity of protein bands after staining: His > Asp >> Arg, Lys, Pro, Glu, Gly, Ser >> Leu
A07. According to Sammons et al. (1983) Anal. Biochemistry, 134, 184-188. Simple procedure for RNA (DNA) staining. Step 01. Wash gel with 50 % ethanol, 10 % acetic acid for 60 min Step 02. Wash gel with 10 % ethanol, 1 % acetic acid for 60 min Step 03. Wash with 12 mM AgNO3 for 60 min Step 04. Rinse quickly with water (10 - 30 sec) Step 05. Development. Wash with 0.28 % formaldehyde, 0.75 M KOH until band appearance Step 06. Wash with 0.07 M Na2CO3 for 10 min Step 07. Wash extensively with water. Comment 1. tRNA is coloured darker if NaOH was used instead of KOH, or if K2CO3 was used instead of Na2CO3 Comment 2. Background staining is high, so that the ratio "signal/noise" is relatively low.
A08. Combined Pyronin/Silver Staining Procedure for RNA (DNA). Optimized for 0.2 - 0.5 mm -thick PAAG slabs. Step 01. Put gel in 0.5 % Pyronin Y, 15 % methanol, 5 % acetic acid, shake gently for 30 min Step 02. Wash gel with 30 % ethanol, 5 % acetic acid, 4 times X 15 min Step 03. Leave gel in 30 % ethanol, 5 % acetic acid, 0.5 ml/l 37 % formaldehyde for at least 1 h (usually - overnight) Step 04. Wash gel with 30 % ethanol, 3 times X 20 min Step 05. Wash gel with 0.2 g/l sodium thiosulfate pentahydrate (Na2S2O3*5H2O), 30 % ethanol for 1 min Step 06. Wash gel with 30 % ethanol, 3 times X 20 sec Step 07. Wash gel with 2 g/l AgNO3, 0.75 ml/l 37 % formaldehyde, 30 % ethanol for 20 min Step 08. Wash gel with 30 % ethanol, 2 times X 20 sec Step 09. Development. Put gel in freshly prepared solution of 60 g/l Na2CO3 (or 162 g/l Na2CO3*10H2O) , 0.5 ml/l 37 % formaldehyde, 4 mg/l sodium thiosulfate pentahydrate (Na2S2O3*5H2O) until band appearance (2 - 5 min) Step 10. Wash gel with water, 2 times X 2 min Step 11. Wash gel with 1 % acetic acid Step 12. Wash gel with water, 2-3 times X 30 min Step 13. Put gel in 5 % glycerol for 1 h, then dry. Comment 1. Sensitivity is about 0.5 - 2 pg of RNA per band Comment 2. Upon development(Step 09), gel increases in size because of transfer from 30 % ethanol to water-based solution. That speeds up diffusion of developer inside the gel Comment 3. Try to reduce pink background after Pyronin staining to minimum, otherwise background staining with silver could be high. Wash gel with extra portions of 30 % ethanol, 5 % acetic acid (Step 2) if necessary Comment 4. Carefully perform wash after sodium thiosulfate treatment (Step 05), large residual amount of sodium thiosulfate may cause high background staining and unequal staining intensity of different parts of the gel Comment 5. Comparison of combined Pyronin/Silver staining with an equivalent procedure without Pyronin treatment reveals a noticeable increase of sensitivity, better contrast, faster band appearance upon development.
A09. According to Marshall et al. (1984) Anal. Biochem., 136, 340-346 Optimized for 0.8 - 3.0 mm -thick PAAG slabs. Step 01. Fixation. Put gel in 50 % methanol, 10 % acetic acid, leave overnight Step 02. Wash gel with water at 60 oC, 3 - 4 times X 5 min Step 03. Wash gel with solution of 2.5 ml/l 37 % formaldehyde at 60 oC for 30 min Step 04. Wash gel with water at room temperature for 10 min Step 05. Prepare staining solution: mix 1 volume of 30 % methylamine with 5 volumes of 0.36 % NaOH, then add 10 ml of this mixture slowly to 4 ml of 20 % AgNO3 upon permanent stirring. At the beginning brown precipitate can be seen, however later solution should become transparent. Adjust volume to 100 ml with water Step 06. Wash gel with staining solution at room temperature for 10 min Step 07. Rinse gel twice with water Step 08. Development. Wash gel with 0.05 mg/ml citric acid, 0.5 ml/l 37 % formaldehyde, up to 6 times X 5 min, until band appearance Step 09. Wash gel with water 2-3 times X 10 min Step 10. Destaining. If background staining was too high, you can destain gel. Dissolve 11.1 g NaCl and 11.1 g CuSO4 in 285 ml H2O, and slowly add 25 % NH4OH (concentrated solution of NH3 in water) upon stirring. At the beginning precipitate will be formed, but later solution turns transparent and dark-blue. Mix 3 volumes of this solution with 1 volume of 44 % sodium thiosulfate, then add 4 volumes of water. Put gel in this solution for 1 - 4 min until background clearance, then transfer it in 44 % sodium thiosulfate for 30 min. Finally wash gel with water, 3 times X 20 min Comment 1. Combination of deep staining with subsequent destaining results in high "signal/noise" ratio. Sensitivity is 10 pg of BSA per mm2
_Dascha_/11.01.2006 21:38/
Я знаю по крайней мере еще 2 метода окрашивания гелей серебром, при чем это подходит не только для SDS-гелей, но и для кислого геля. Метод 1 взят из Wray W., Boulikas T., Wray V.P. and Hancock R. 1981. Silver staining of proteins in polyacrylamide gels. Anal Biochem 118-197. Итак: 1. Перенести гели в чистые контейнеры (в которых не окрашивали гели с помощью Coumassie). Все этапы необходимо проводить при постоянном помешивании (на шейкере). Исходные растворы: 0.36% (w/v) NaOH 20% (w/v) AgNO3 1.05% citric acid Сама процедура 1. Промыть каждый гель в 50% метаноле 5 раз по 15 минут 2. Приготовить следующие растворы (каждый раз свежие) для окрашивания серебром. На 2-3 геля silver nitrate solution: 21.0 ml 0.36% NaOH 2.0 ml ammonium hydroxide 77.0 ml dH2O Помешивая, добавляйте 20% AgNO3 до тех пор, пока раствор не станет темно-коричневым. После этого надо добавить 1-2 капли гидроксида аммония и раствор снова станет прозрачным. developing solution 0.5 ml 1.05% citric acid 50.0 мкл 37% (v/v) formaldehyde 99.5 ml dH2O stop soln 50 ml methanol 10.0 ml lacial acetic acid 40.0 ml H2O 3. Заменить метанол, которым промывались гели на раствор нитрата серебра, держать в нем 15 минут 4. Промыть дист. водой 5 раз по 3 минуты каждый 5. Заменить воду developing solution. Когда оптимальная окраска будет достигнута, надо немедленно промыть дист. водой и быстро заменить на stop soln. 6. Гель должен находиться в stop soln. не менее 10 минут. 7. Высушите гель или замените stop soln. на дист. воду, чтобы избежать обесцвечивания краски.
_Swetik_/11.01.2006 22:13/
Еще одна методика окраски геля серебром: 1. Фиксировать 20 мин 20% ТХУ, затем сполоснуть Н2О сразу и еще 2 раза по 10 мин. Оставить отмываться в Н2О на ночь; 2. Еще 4 раза по 10 мин Н2О; 3. 4-5 мг/мл ДТТ в 300 мл Н2О 30 мин; 4. 0,1% раствор AgNO3 (150 мл) 30 мин (подальше от света); 5. Ополоснуть Н2О 1-4 раза; 6. Ополоснуть 2 раза по 100 мл проявителя (9г Na2CO3 + 300 мл Н2О + 140мкл формалина (формалин добавляют перед самым окрашиванием)); 7. Оставить в 100 мл проявителя до момента окрашивания (занимает около 10-15 мин, зависит от концентрации белка); 8. Нейтрализовать 2 % раствором СH3COOH (100 мл) 10 мин; 9. Сполоснуть гель 2-3 раза Н2О и оставить в ней на несколько часов. 10. Перед тем как сушить (если понадобиться) вымочить гель в течение суток в 8% растворе глицерина.
Комментарии аналогичные аммонийному методу окрашивания.
Гость/04.04.2006 17:33/
Я в лаборатории делаю следующим образом:
biopaul/04.04.2006 18:24/
Так вот: 1. Гель достаем из слаба, кладем в 1% спирт, греем в микроволновке до интенсивного кипения. 2. 10 мин на качалке 3. Кладем в серебряно-нитратный реактив на 15мин (50мл воды 1мл аммиака, 2мл 20% серебра, 2мл 5М NaoH) 4. Моем в воде 3 раза по 5 мин (важно чтобы не было бэкграунда) 5. Кладем в проявляющий раствор до появления полос. Все. Чувствительность не менее 20нг/band
Masha11/09.08.2006 19:56/
Подскажите, пожалуйста, зачем сначала помещать гели в тиосульфат натрия? как проявитель это понятно почему он используется. но вот до окраски серебром не могу понять зачем он добавляется.
Mac-1/10.08.2006 10:13/
Фумара/04.11.2008 17:14/
Скажите, а для окрашивания ДНК эти методики тоже подойдут?
guest: ммм /05.11.2008 12:37/
--Скажите, а для окрашивания ДНК эти методики тоже подойдут?
Нет. ДНК красят чистым нитратом(с кислой реакцией), без всяких гидрокси амонийных комплексов.
Фумара/05.11.2008 15:00/
А вы не могли бы написать полный протокол для окрашивания ДНК?
Гость/19.11.2008 14:25/
Может быть кто-нибудь знаком с тонкостями окраски серебром липоолигосахаридов?
Alenka-s/05.08.2009 15:48/
(Гость @ 19.11.2008 14:25)
Может быть кто-нибудь знаком с тонкостями окраски серебром липоолигосахаридов?
моя методика окрашивания ЛПС (Fomsgaard A. et al,1990), но я по ней красу гликопротеины. 1. Гель без предварительной фиксации выдурживают в растворе №1 (HJO4 - 3,5 г, C2H5OH - 208 мл, конц.уксусная кислота - 25 мл, дист вода- доводим до 500 мл) на протяжении 20 минут. 2. Гель отмывают 3 раза по 5 минут в дист. воде. 3. Выдерживаем гель на протяжении 10 минут в свежеприготовленном растворе № 2 (0,1 М NaOH - 56 мл и конц. NH4OH - 1 мл перемешиваем на мешалке, добавляем туда 100 мл дист. воды и по каплям 20 % р-р AgNO3 - 10 мл, полученый раствор доводим до 300 мл дист. водой). 4. Гель отмываем 3 раза по 5 минут в дист. воде. 5. Проявляем гель раствором № 3 (лимонная кислота - 10 мг, 37 % формальдегид - 0,1-0,2 мл, дист. вода - 200 мл). 5. Остановка реакции - 10 % уксусной кислотой
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