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p2011

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lstt09nk(½ð±Ò+2): ¸Ðл²ÎÓë 2011-04-13 19:41:01
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Originally posted by p2011 at 2011-04-12 15:00:22:
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p2011

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Originally posted by jhu132llh at 2011-04-12 15:46:21:
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reasonspare

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lstt09nk(½ð±Ò+5): »¶Ó­»Ø¹é^_^ 2011-04-13 19:41:39
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Originally posted by p2011 at 2011-04-12 16:16:38:
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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 ¦Ìl 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 ¦Ìl formalin (35% Formaldehyde)
200 ml H2O
Developer: 6% Na2CO3, 0.05% formalin (35% Formaldehyde),
0.0004% Na2S2O3 400 ml
24 g Na2CO3
200 ¦Ìl 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.
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reasonspare

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   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)
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7Â¥2011-04-16 10:50:17
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xhjggl

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