AMIDO BLACK STAIN 氨基黑染色蛋白

• 250 ml water
• 200 ml methanol
• 50 ml acetic acid
• 0.1% (w/v) Amido Black (also called Napthol Blue Black)

• 800 ml methanol (20%)
• 300 ml acetic acid (7.5%)
• 2900 ml distilled water (72.5%)
  
  LPS SILVER STAIN #1
  
  REFERENCE: Hitchcock and Brown. (1983). Journal of Bacteriology, 154:269-277.
  
  OBJECTIVE:
  
  To stain whole cells LPS gels.
  
  METHOD:
  
  1. After running the gel, fix overnight in 25% (v/v) isopropanol in 7% acetic acid. You do not need to use the rotary shaker.
     
  2. The next morning, oxidize the gel for 5 minutes with 2.1g periodic acid and 8ml of isopropanol in 300 ml of distilled water. Make this solution fresh before using.
     
  3. Wash the gel 8 x 30 minutes in distilled water at room temperature on the rotary shaker.
     
  4. Incubate for 10 minutes with silver stain on the rotary shaker.
     
     

     SILVER STAIN:

     56 ml 0.1N NaOH

     230 ml distilled water

     2 ml 29.4% ammonium hydroxide

     Add 9.8ml of 20% silver nitrate solution slowly with shaking. Make this solution fresh right brfore using. It is written that you can dissolve 10 ml of the silver solution but I have found that in fact you can dissolve only 9.8. It is very important to keep the ammonium hydroxide tightly covered as its strength decreases rapidly if left open even for a short time.

     
     
     
     
  5. 4 X 10 minute washes with distilled water on the shaker.
     
  6. 10 to 20 minutes in the developer.
     
     

     DEVELOPER:

     50 mg citric acid

     0.5 ml 37% formaldehyde

     1 litre distilled water

     Make fresh before use. Also raise the temperature of this solution to above 26?C to help preferentially stain the LPS.

     
     
     
  7. Incubate the gel for 1 hour in a stop bath consisting of 20 ml of 7% acetic acid in 400 ml of distilled water.
     
  8. Final wash in distilled water for one hour. After this point you can store the gel in water but taking a photograph immediately prevents any deterioration which might occur.
  
  
  NOTES:
  
  1. Be sure to clean all glassware well and change glass dishes between the wash after the silver is added and the addition of the developer.
     
  2. If you use gloves rinse them well in distilled water before use to eliminate contamination with talc.
  
  
  LPS SILVER STAIN #2
  
  REFERENCES: Tsai and Frasch. 1982. Anal. Biochem. 119:115.
  
  Preferred method for LPS gel staining
  
  METHOD:
  
  1. Soak gel overnight in 40% ethanol (for outer membrane or cell envelopes, subsitute isopropanol for ethanol to amplify LPS) and 5% acetic acid in 200 ml of distilled water.
     
  2. Soak the gel for 5 minutes in periodic acid solution to oxidise the LPS.
     
     

     0.7% periodic acid	1.4 g
     40% ethanol (or isopropanol)	84.2 ml
     5% acetic acid	10.0 ml
     Make up to 200 ml with distilled water	?

     
     
     
  3. Rinse in running distilled water for 2 hours.
     
  4. Put developer in the 37? room to warm up.
     
  5. Add staining reagent and agitate for 10 minutes.
     
     

     2 ml concentrated ammonium hydroxide (use fresh frozen stock)

     28 ml 0.1N NaOH

     5 ml 20% silver nitrate (add dropwise to above with vigorous stirring)

     115 ml distilled water

     
     
     
  6. Rinse and wash 3 times in distilled water (15 min/wash).
     
  7. Change to a new dish and add warm developer.
     
     

     10 mg citric acid

     0.1 ml 30% formaldehyde

     200 ml distilled water

     
     
     
  8. Stop the staining reaction by reaction by washing with 0.35% acetic acid (1.4 ml glacial acetic acid in 400 mls dH2O). Fix in this solution for at least one hour. Store in distilled water.
     
  9. Destain as for protein silver stain.
  
  
  NOTES:
  
  Be sure to clean all glassware well and change glass dishes between the wash after the silver is added and the addition of the developer.
  

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