STAINS
 

AMIDO BLACK STAIN
Use to stain protein on nitrocellulose blots.

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


DESTAINING SOLUTION FOR POLYACRYLAMIDE GELS

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.

    5. 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.

    7. 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 26ºC 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 watter.
  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.

    3. 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 37º room to warm up.
  5. Add staining reagent and agitate for 10 minutes.

    6. 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.

    8. 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.
If you use gloves rinse them well in distilled water before use to eliminate contamination with talc.


PROTEIN SILVER STAIN

MATERIALS:

Stain:
1.4 ml Ammonium hydroxide (use the fresh, frozen stock aliquoted in the -20ºC freezer)
21.0 ml NaOH (0.36%)
4.0 ml AgNO3 (20% w/v) added dropwise
distilled water to 100 ml

Developer:
2.5 ml citric acid (1% w/v)
0.25 ml formaldehyde (38% v/v)
distilled water to 250 ml

METHOD:

  1. Soak the gel overrnight in 50% methanol.
  2. Add fresh 50% methanol and leave 1 hour.
  3. Wash briefly in distilled water.
  4. Agitate for 10 minutes in staining reagent.
  5. Rinse briefly in distilled water and soak for 5 minutes in fresh distilled water.
  6. Rinse again briefly in distilled water. Be careful not to let the gel get stuck to the glass dish at any point after staining step. Change to a new dish.
  7. Add developer and agitate for 5 to 10 minutes.
  8. When the gel achieves the desired state of staining, soak it in 50% methanol/10% acetic acid overnight to stop developing.
  9. Photograph the gel before drying in case it cracks or darkens.


DESTAINING:

If necessary, the gel may be destained using a 1:4 dilution of Kodafix (the photographic stuff) in 5 % Methanol. This slows the destaining, so don't go overboard.

To stop destaining use a 1:31 dilution of Hypo Eliminator (the photographic stuff). When destaining is complete, the gel must be soaked in 50% methanol before restaining.


Updated 01 December 2000