Mass Spectrometry Facility - Gel StainingDestaining Protocols

The following protocols are gel staining methods that are compatible with mass spectrometry.

• Coomassie Brilliant Blue Staining [original link path: https://www.artsci.uc.edu/departments/chemistry/core-facilities/mass-spectometry-facility/mass-spectometry-facility---protocols/mass-spectometry-facility---gel-staining-destaining-protocols.html]
• Zn-imidazole Reverse Staining [original link path: https://www.artsci.uc.edu/departments/chemistry/core-facilities/mass-spectometry-facility/mass-spectometry-facility---protocols/mass-spectometry-facility---gel-staining-destaining-protocols.html]
• Copper Reverse Staining [original link path: https://www.artsci.uc.edu/departments/chemistry/core-facilities/mass-spectometry-facility/mass-spectometry-facility---protocols/mass-spectometry-facility---gel-staining-destaining-protocols.html]
• Vorum Modified MS Silver Staining [original link path: https://www.artsci.uc.edu/departments/chemistry/core-facilities/mass-spectometry-facility/mass-spectometry-facility---protocols/mass-spectometry-facility---gel-staining-destaining-protocols.html]
• EMBL Silver Staining [original link path: https://www.artsci.uc.edu/departments/chemistry/core-facilities/mass-spectometry-facility/mass-spectometry-facility---protocols/mass-spectometry-facility---gel-staining-destaining-protocols.html]

Buffers

• Fixer: 45.4 %MeOH, 4.6 %HAc, vol. 1000 ml.
• 454 ml MeOH
• 454 ml H2O
• 92 ml HAc

• Stain: 45.4 %MeOH, 4.6 %HAc, 0.1 %Coomassie, vol. 1000 ml.
• 454 ml MeOH
• 454 ml H2O
• 92 ml HAc
• 1 g Coomassie Brilliant Blue R-250
• Filter solution before use!

• Destain: 5 %MeOH, 7.5 %HAc, vol. 1000 ml.
• 50 ml MeOH
• 875 ml H2O
• 75 ml HAc

Procedure

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

Buffers

• Stain: 0.2 M imidazole, 0.1% SDS, pH 8.0, vol. 200 ml.
• Stain: 0.2 M Zn-sulphate or 0.2 M ZnCl2 (pH 2.5), vol. 200 ml.
• Wash: MilliQ water, vol. 200 ml.

Procedure

1. Wash gel
   Water for 30-60 sec.
2. Soak gel
   0.2 M imidazole, 0.1% SDS for 15 mins.
3. Stain gel
   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
   3 washes with water.
5. Mobilize protein
   0.5 ml 50 mM NH4HCO3, 100 mM DTT for 10-20 min, until the gel plug is completely transparent.

See primary reference: C. Fernandez-Patron et al. Anal. Biochem. 224, 203-211, 1995 for more information.

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

• Fixer: 50% MeOH, 12% HAc, 0.05% formalin (35% Formaldehyde), 200 ml
  100 ml MeOH (99.8%)
  24 ml HAc (100%)
  100 ml formalin (35% Formaldehyde)
  76 ml H2O (Milli-Q)
• 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% AgNO3, 0.076% formalin (35% Formaldehyde), 200 ml
  0.4 g AgNO3
  152 ml formalin (35% Formaldehyde)
  200 ml H2O
• Developer: 6% Na2CO3, 0.05% formalin (35% Formaldehyde), 0.0004% Na2S2O3, 400 ml
  24 g Na2CO3
  200 ml formalin (35% Formaldehyde)
  8 ml 0.02% Na2S2O3
  392 ml H2O
• Stop Solution: 50% MeOH, 12% HAc, 200 ml
  100 ml MeOH
  24 ml HAc
  76 ml H2O

Procedure

1. Fix gel with fixer solution for 2 hrs or overnight
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 for 20 mins
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 for 5 mins
9. Leave the gel at 4 ?C in 1% acetic acid

Buffers

• Fixer: 50% methanol, 5% acetic acid, vol. 200 ml.
  100 mL methanol
  10 mL acetic acid (100%)
  90 mL Milli-Q water
• Wash: 50% methanol, vol. 200 mL
  100 mL methanol
  100 mL Milli-Q water
• Sensitizing: 0.02% Na2S2O3, vol. 200 mL
  0.04 g Na2S2O3
  200 mL Milli-Q water
• Silver Nitrate: 0.1% AgNO3, vol. 200 mL (Cold)
  0.2 g AgNO3
  200 mL Milli-Q water
• Developer: 0.04% Formalin, 2% Na2CO3, vol. 250 ml
  100 mL Formalin (35% Formaldehyde)
  5 g Na2CO3
  250 mL Milli-Q water
• Stop: 5% acetic acid, vol. 200 mL
  10 mL acetic acid (100%)
  190 mL Milli-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

See primary reference: A. Schevchenko et al. Anal. Chem, 68, 850-858, 1996 for more information