Staining of Gels for Mass Spectrometric Analysis

Coomassie Brilliant Blue staining

Buffers
 Fixer:İ45.4%İMeOH, 4.6%İHAc, vol. 1000 ml.
  454 ml MeOH
  454 ml H₂O
  92 ml HAc

 Stain:İ45.4%İMeOH, 4.6%İHAc, 0.1%İCoomassie, vol. 1000 ml.
  454 ml MeOH
  454 ml H₂O
  92 ml HAc
  1 g Coomassie Brilliant Blue R-250
  Filter solution before use!

 Destain:İ5%İMeOH, 7.5%İHAc, vol. 1000 ml.
  50 ml MeOH
  875 ml H₂O
  75 ml HAc

İ Procedure

1. Fix gel
   İİ 100 ml 45.4% MeOH, 4.6% HAc for 1 hour. Do not fix gels for preparative use
   
2. Stain gel
   İİ 100 ml 45.4% MeOH, 4.6% HAc, 0.1% Coomassie for 1 hour.
   
3. Destain gel
   İİİİ 100 ml 5% MeOH, 7.5% HAc for 24 hours. Replace if needed.

Zn-imidazole reverse staining

İ Procedure

1. Wash gel
   İİ Water for 30-60 sec.
   
2. Soak gel
   İİ 0.2 M imidazole, 0.1% SDS for 15 mins.
   
3. Stain gel
   İİ 0.2 M Zn-sulphate or 0.2 M ZnCl₂ for approx. 30-60 secs.until the gel background becomes white leaving transparent protein bands.
   
4. Stop staining
   İİ 3 washes with water.
   
5. Mobilize protein
   İİİİ 0.5 ml 50 mM NH₄HCO₃, 100 mM DTT for 10-20 min, until the gel plug is completely transparent.

Copper reverse staining

Buffers
 Stain: 0.3 M CuCl₂, vol. 100 ml.
  4 g CuCl₂
  100 ml MilliQ water

 Wash: MilliQ water, vol. 200 ml.

Procedure

1. Wash gel
   İİ Water for 30-60 sec.
   
2. Stain Gel
   İİ 0.3 M CuCl₂ for 5 minutes while rocking
   
3. Stop staining
   İİ 3 washes with water.
   

Vorum modified MS Silver staining - Our facility's first choice among the silver stains

 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%İAgNO3, 0.076%İformalinİ(35%İFormaldehyde), 200 ml
  0.4 g AgNO₃
  152 ml formalin (35%İFormaldehyde)
  200 ml H₂O

 Developer: 6%İNa₂CO₃, 0.05%İformalinİ(35%İFormaldehyde), 0.0004%İNa₂S₂O₃, 400 ml
  24 g Na₂CO₃
  200 ml formalinİ(35%İFormaldehyde)
  8 ml 0.02% Na₂S₂O₃
  392 ml H₂O

 Stop solution: 50%İMeOH, 12%İHAc, 200 ml
  100 ml MeOH
  24 ml HAc
  76 ml H₂O

Procedure

1. Fix gel with fixer solution for 2İhrsİorİovernight
2. Wash gel with wash solution for 20 mins (repeat 3 times)
3. Sensitize gel with sensitizing solution for 2 mins
4. Wash gel with Milli-Q water for 5 mins (repeat 3 times)
5. Stain gel with silver nitrate solution forİ20İmins
6. Wash gel with Milli-Q water for 1 min (repeat 2 times)
7. Develop gel with developing solution
8. Stop staining with stop solution forİ5İmins
9. Leave the gel at 4 †C in 1% acetic acid

EMBL Silver staining

 Wash: 50%İmethanol, vol.İ200İmL
  100İmLİmethanol
  100İmLİMilli-Q water

 Sensitizing: 0.02%İNa₂S₂O₃, vol.İ200İmL
  0.04İgİNa₂S₂O₃
  200İmLİMilli-Q water

 Silver nitrate: 0.1%İAgNO₃, vol.İ200 mLİ(Cold)
  0.2İgİAgNO₃
  200İmL Milli-Q water

 Developer: 0.04%İFormalin, 2%İNa₂CO₃, vol.İ250 ml
  100İmLİFormalin (35%İFormaldehyde)
  5İgİNa₂CO₃
  250İmL Milli-Q water

 Stop: 5% acetic acid, vol.İ200İmL
  10İmL acetic acidİ(100%)
  190İmLİMilli-Q water

Procedure

1. Fix gel with fixing solution for 20 mins
2. Wash gel with wash solution for 10 mins
3. Wash gel with Milli-Q water for 2 hrs
   Reduce background staining by over night washing.
   
4. Sensitize gel with sensitizing solution for 1 min
5. Wash gel with Milli-Q water for 1 min (repeat 2 times)
   
6. Incubate gel in cold silver nitrate solution for 20 mins at 4 †C
7. Wash gel with Milli-Q water for 1 min
   
8. Change gel chamber
9. Wash gel with Milli-Q water for 1 min
   
10. Develop gel with developer solution
    Observe the color and change solution when the developer turns yellow. Terminate when the staining is sufficient.
    
11. Terminate developing with stop solution
    Change the solution a couple of times
    
12. Leave the gel at 4 †C in 1% acetic acid