Staining Protocols

These protocols are intended as general guides only. We suggest you use a staining kit from one of the major vendors. If using the protocols below please test them using standard controls prior to using them on your samples. Prepare all solutions the same day you will stain the gel.

Standard Coomassie Brilliant Blue Staining

Sensitivity ~ 100 ng

  1. Rinse gel in water sufficient to cover gel, 10 min, repeat 2x
  2. Stain gel using 0.1% CBB R-250, 20% methanol, 0.5% acetic acid, 30 min to overnight
  3. Discard stain and wash gel in water, 10 min, repeat
  4. Destain in 30% methanol
  5. Wash gel in water, 10 min, repeat

High-sensitivity Coomassie Brilliant Blue Staining

Sensitivity ~ 10 ng

0.02% (w/v) CBB G-250
[40 mg]
5% (w/v) aluminum sulfate-(14-18)-hydrate
[10 g]
10% (v/v) ethanol
[20 mL]
2% orthophosphoric acid (85%)
[4.7 mL]

*add components in order, otherwise sensitivity will decrease: dissolve aluminum sulfate in water, then add ethanol, mix, then add CBB, mix, then add orthophosphoric acid, mix, add water to 200 mL

  1. Rinse gel in water sufficient to cover gel, 10 min, repeat 2x
  2. Stain gel 2 hr to overnight
  3. Discard stain, wash gel in water, 10 min, repeat
  4. Destain gel 10-60 min in 10% ethanol, 2% orthophosphoric acid
  5. Wash gel in water, 10 min, repeat

Silver Staining

Sensitivity ~ 1 ng

  1. Place gel in fixing solution (40% ethanol, 10% acetic acid) 1 h
  2. Rinse gel in water 1 h – overnight
  3. Place gel in sensitizing solution (0.02% sodium thiosulfate) 1 m ONLY!
  4. Wash gel in water, 30 s, repeat 2x
  5. Place gel in cold (4 C) silver stain solution (0.1% silver nitrate, 0.02% formaldehyde)
  6. Wash gel in water, 30 s, repeat 2x
  7. Place gel in developer using new, clean staining dish, 3% sodium carbonate, 0.05% formaldehyde)
  8. Observe gel carefully, staining can proceed very quickly and it is easy to overstain
  9. Wash gel in water, 30 s
  10. Fix gel in 5% acetic acid