Coomassie and Silver Staining of Polyacrylamide Gels
Prepare and electrophorese protein samples on polyacrylamide gels (see gel preparation protocol for large gels, or Western blot protocol for small gels). Protein sample preparation procedures are listed in the Western blot protocol.

For optimal visualization of proteins on Coomassie blue stained gels, additional protein needs to be loaded relative to that used for Western blots. For bacteria, use about five times more, and for insect cell extracts use about ten times more protein.


COOMASSIE STAINING
Coomassie Blue staining is reported to be 3 times more sensitive than Fast Green and 6 more sensitive than Amido Black. Coomassie blue is used to stain the gel, not a Western blot membrane.

  1. Disassemble the gel apparatus and transfer the polyacrylamide gel to a glass tank.
  2. Cover with Coomassie Gel Stain for 3 hours.
  3. The gel is destained from about 4 hours to overnight. Change the destaining solution 3 - 4 times. The gel can be re-swollen in H2O. Dry gel for 1 hour on the gel dryer set from 60 - 80°C.

SILVER STAINING
(Adapted from the Bio-Rad Kit Protocol #LIT-34 89-0559 689; additional comments from Morrissey, 1981. Anal. Biochem. 117:307-310.

Handling the gel
Wear gloves and use glass containers. Everything that comes in contact
with the gel must be clean including electrophoresis plates and gloves. Touch the gel or glass with your fingers typically leaves a fingerprint after staining. Rinse everything in deionized water after washing, and where appropriate, clean with methanol. Bio-Rad recommends the use of deionized water with conductivity below 1 mmho.

When transferring the gel to a staining dish and when transferring solutions, do not apply pressure to the gel. Stain each gel in a separate container. We perform each step on a slowly rotating orbital shaker (50-60 rpm) at room temperature and use enough of each solution to submerse the gel and allow its free movement.

The following time intervals are appropriate for a 0.5 - 1.0 mm thick polyacrylamide gel. Prepare the developer fresh each day. Transfer the gel to fixative #1 immediately after electrophoresis. The gel may be stored indefinitely in this fixative prior to staining.

1. Fixative 1 30 min
2. Fixative 2 15 min
3. Fixative 2 15 min
4. Oxidizer (dilute to 1X before use) 5 min.
5. Deionized Water 5 min.
6. Deionized Water 5 min.
7. Deionized Water until all yellow color is removed from gel
8. Silver Reagent (dilute to 1X before use) 20 min.
9. Deionized Water 1 min
10. Developer (freshly made) about 30 sec. Replace when a smoky brown precipitate appears
11. Developer about 5 min.
12. Developer (optional) about 5 min.
13. Stop solution 5 min. or over-night

The developing steps must be closely watched for optimal staining. A cloudy precipitate forms rapidly during the first stage (stop 10), but much more slowly in the second stop. The final development must be watched to avoid over staining the gel. It may not be necessary, depending on the staining intensity at the end of the second developing step (step 11). Higher molecular weight proteins tend to stain before lower ones. At some point in the final developing step, the background intensity will increase at about the same rate as the band intensity. Stop the reaction at this time to avoid dark grey backgrounds.

Photographing Gels
After stopping the development, gels may be photographed on a light box (with illumination from below). When using type 57 Polaroid film, f32 and 1/125 second is recommended. Gels may be photographed through a Wratten No. 45 filter.

Destaining
A high background of silver on overstained gels may be partially corrected using the method of Switzer, et al, Anal. Biochem. 98, 231 (1979).
    Destain Solution 1
    Dissolve 37 g of sodium chloride and 37 g of cupric sulfate anhydrous in 850 ml of deionized water. Add concentrated ammonium hydroxide until all of the precipitate is dissolved. Q.S to 1 L with deionized water.
    Destain Solution 2.
    Dissolve 436 g sodium thiosulfate pentahydrate in 1 liter of deionized water. Destain with a freshly prepared solution consisting of equal volumes of destain solutions 1 and 2 immediately prior to use. Do not completely destaining the gel. Wash with deionized water to remove the destain so0lution and prevent fogging. Stop the reaction by soaking the gel in 10% acetic acid for about 15 minutes. Then extensively wash the gel for at least one hour with a number of water changes. Restaining can be carried out beginning with the addition of fresh silver reagent.

TROUBLE SHOOTING
 Gray or brown precipitate appearing as smudges or swirling on gel surface. Bands may be faint or absent.
  1. Non-specific deposition of silver due to oxidizer or silver reagent carry-over.  
  2. Increase wash steps. (Increase steps 5, 6, & 7 to 20 min. each.
  3. Temperature is too low. Make sure temperature of reagents is at least 23°C.
  4. The developer precipitate sticking to the gel surface can cause mirroring.
  5. Be sure to decant first development solution as soon as the precipitate appears.
  6. May be due to different states of reduction of proteins from different tissues.  
  7. Reducing proteins with dithiothreitol according to Morrissey may help.
Dark uniform background, usually yellow.
  1. Oxidizer is not completely removed. Increase wash steps 5, 6, and 7 as described above, removing all traces of yellow, before going on to silver reagent step.
Mottled background, usually brown or green, with poor sensitivity.  
  1. Contaminants in water. Check that the conductivity of the water is less than 1 mmho.
  2. Incomplete removal of gel buffer components. Increase times of steps 1, 2, 3 to ensure complete removal.
Slow or no development
  • Rate of development is highly temperature
  • Spotty background
  • Dust particles in solution. Filter solutions through Millipore filters.

    • RECIPES

    Coomassie Staining
      0.2% Coomassie Gel Stain (in 7.5% acetic acid and 40% methanol):
      0.2 g Coomassie
      7.5 ml acetic acid
      40 ml methanol
      Q.S. with H2O to 100 ml. Nalgene or Whatman filter before use.

      Coomassie Rapid Destain (7.5% acetic acid and 5% methanol):
      75 ml acetic Acid
      50 ml methanol
      Q.S. with H20 to 1 L.

    Silver Staining (Charles O'Brien)
      Fixative 1
      40% methanol
      10% acetic acid
      Prepare 500 ml

      Fixative 2
      10% ethanol
      5% acetic acid
      Prepare 1L

      10x Oxidant
      2.5 grams potassium dichromate (0.034M)
      500 ml nitric acid (0.032M)
      QS to 250 ml with double distilled water. Store at 4°C for up to one year. Make sure the working dilution is at room temperature before use.

      10x Silver Nitrate
      5.10 grams silver nitrate (0.12M)
      QS to 250 ml with double distilled water. Store at 4ºC for up to one year. Make sure the working dilution is at room temperature before use.

      Developer (make fresh each time)
      29.68 grams sodium carbonate (0.28M)
      0.5 ml formalin (formaldehyde)
      QS to one liter with double distilled water. Make sure the solution is at room temperature before use.

      Stop solution
      5% acetic acid

    Protein Molecular Weight Markers
    We have used Promega and Pharmacia molecular weight markers.
    Promega Mid-Range Pharmacia LMW
    Phosphorylase B 97.4 kD Phosphorylase B 94 kD
    BSA 66.2 kD BSA 67 kD
    L-glutamate dehydrogenase 55 kD  
    ovalbumin 42.7 kd ovalbumin 43 kD
    aldolase 40 kD  
    carbonic anhydrase 31kD carbonic anhydrase 30 kD
    soybean trypsin inhibitor 21.5 kD trypsin inhibitor 22 kD
    lysozyme 14.3 kd a-lactalbumin 14.4 kD


        Send comments and updates to  Dr. Bart Frank, Arthritis and Immunology Program, OMRF
    Return to Protocols: Table of Contents
    Return to the Frank Lab Home Page
    Copyright 1997 and 1998 by Mark Barton Frank, Ph.D.
    Proper citation for data acquired from this document is: "Frank, M. B. Coomassie and Silver Staining of Polyacrylamide Gels. In: Frank, M. B. ed. Molecular Biology Protocols. (http://omrf.ouhsc.edu/~frank/depc.html). 1997. Oklahoma City. Revision Date: September 15, 1998."