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【求助】銀染法的具體步驟 已有10人參與
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jhu132llh
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reasonspare
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lstt09nk(金幣+5): 歡迎回歸^_^ 2011-04-13 19:41:39
小木蟲(金幣+0.5):給個紅包，謝謝回帖交流
lstt09nk(金幣+5): 歡迎回歸^_^ 2011-04-13 19:41:39
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Silver staining protocol Compatible with mass spectrometry, excellent signal-to-noise ratio (Ref: Protana website http://www.protana.com/services/protocols/default.asp) Buffers Fixer: 50% MeOH, 12% HAc, 0.05% formalin (35% Formaldehyde) 200 ml 100 ml MeOH (99.8%) 24 ml HAc (100%) 100 μl formalin (35% Formaldehyd) 76 ml H2O (Milli-Q) Wash: 35% EtOH (96%) 200 ml 73 ml EtOH 127 ml H2O Sensitizing: 0.02% Na2S2O3 200 ml 0.04 g Na2S2O3 200 ml H2O Silver nitrate:0.2% AgNO3, 0.076% formalin (35% Formaldehyde) 200 ml 0.4 g AgNO3 152 μl formalin (35% Formaldehyde) 200 ml H2O Developer: 6% Na2CO3, 0.05% formalin (35% Formaldehyde), 0.0004% Na2S2O3 400 ml 24 g Na2CO3 200 μl formalin (35% Formaldehyde) 8 ml 0.02% Na2S2O3 392 ml H2O Stop solution:50% MeOH, 12% HAc 200 ml 100 ml MeOH 24 ml HAc 76 ml H2O Procedure 1. Fix gel: 50% MeOH, 12% Hac, 0.05% formalin, 2 hrs or overnight 2. Wash gel: 35% EtOH for 20 mins. 3. Wash gel: 35% EtOH for 20 mins. 4. Wash gel: 35% EtOH for 20 mins. 5. Sensitize gel: 0.02% Na2S2O3 for 2 mins. 6. Wash gel: H2O for 5 mins. 7. Wash gel: H2O for 5 mins. 8. Wash gel: H2O for 5 mins. 9. Stain gel: 0.2% AgNO3, 0.076% formalin for 20 mins. 10. Wash gel: H2O for 1 min. 11. Wash gel: H2O for 1 min. 12. Develop gel: 6% Na2CO3, 0.05% formalin, 0.0004% Na2S2O3 13. Stop staining: 50% MeOH, 12% HAc for 5 mins. 14. Leave the gel at 4ºC in: 1% Hac Remark: For coomassie blue stained gel, wash O/N in water, then start from step 5. |
6樓2011-04-13 10:59:21
reasonspare
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小木蟲(金幣+0.5):給個紅包，謝謝回帖交流
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可是我的是核酸步驟同蛋白的銀染步驟相同嗎？？謝謝.. 步驟不是完全一致的， 具體如下 1. Separate plates while keeping the gel attached to short glass. 2. Fix the gel: Place gel in tray, cover with cold fix/stop solution and agitate well for 20 minutes. Gel may be stored in fix/stop solution overnight. Save fix/stop solution and place back in freezer. 3. Wash the gel: Rinse the gel 3 times for 2-3 min. each in ddH2O using agitation. Lift gel from solution and allow to drain 10-20 seconds. 4. Stain the gel: Transfer the gel to staining solution and agitate well for 30 minutes. 5. Pour 1L of the developing solution into a tray. Transfer staining solution to beaker. Rinse tray and fill with ddH2O. 6. Rinse gel for 5-10 seconds ONLY. Transfer to developing solution. 7. Agitate in developing solution until bands begin to appear. Transfer gel to remaining chilled developing solution for 2-3 minutes. 8. Fix the gel: add 1L of Fix/stop solution directly to developing solution and agitate for 2-3 minutes 9. Rinse gel twice for two minutes each in ddH2O. 10. Dry gel on glass Fix/stop solution Staining solution 200ml of glacial acidic acid 2g (1 packet) of silver nitrate (AgNO3) 1,800ml ultrapure water 3ml (1 vial) of 37% Formaldehyde Freeze for approx. 3 hours 2L ultrapure water Developing solution 60g (1 packet) Sodium Carbonate (Na2CO3) 2L ultrapure water **chill to 10oC. I place sol. In freezer for approx 4 hours and stir to break up ice prior to use. Immediately before use add 3ml (1 vial) of 37% Formaldehyde 400m l aliquot Sodium Thiosulfate (discard remaining) |
7樓2011-04-16 10:50:17
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8樓2012-08-02 20:01:52
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silicare: 金幣-5, 應(yīng)助指數(shù)-2, 屏蔽內(nèi)容 2012-08-31 18:30:36
小木蟲: 金幣+0.5, 給個紅包，謝謝回帖
送鮮花一朵silicare: 金幣-5, 應(yīng)助指數(shù)-2, 屏蔽內(nèi)容 2012-08-31 18:30:36
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9樓2012-08-06 13:11:10
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