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]
Coomassie Brilliant Blue Staining
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
- Fix gel
100 ml 45.4% MeOH, 4.6% HAc for 1 hour. Do not fix gels for preparative use - Stain gel
100 ml 45.4% MeOH, 4.6% HAc, 0.1% Coomassie for 1 hour. - Destain gel
100 ml 5% MeOH, 7.5% HAc for 24 hours. Replace if needed.
Zn-imidazole Reverse Staining
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
- Wash gel
Water for 30-60 sec. - Soak gel
0.2 M imidazole, 0.1% SDS for 15 mins. - 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. - Stop staining
3 washes with water. - 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.
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
- Wash gel
Water for 30-60 sec. - Stain Gel
0.3 M CuCl2 for 5 minutes while rocking - Stop staining
3 washes with water.
Vorum Modified MS Silver Staining - Our facility's first choice among the silver stains Buffers
- 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
- Fix gel with fixer solution for 2 hrs or overnight
- Wash gel with wash solution for 20 mins (repeat 3 times)
- Sensitize gel with sensitizing solution for 2 mins
- Wash gel with Milli-Q water for 5 mins (repeat 3 times)
- Stain gel with silver nitrate solution for 20 mins
- Wash gel with Milli-Q water for 1 min (repeat 2 times)
- Develop gel with developing solution
- Stop staining with stop solution for 5 mins
- Leave the gel at 4 ?C in 1% acetic acid
EMBL Silver Staining
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
- Fix gel with fixing solution for 20 mins
- Wash gel with wash solution for 10 mins
- Wash gel with Milli-Q water for 2 hrs. Reduce background staining by over night washing.
- Sensitize gel with sensitizing solution for 1 min
- Wash gel with Milli-Q water for 1 min (repeat 2 times)
- Incubate gel in cold silver nitrate solution for 20 mins at 4 ?C
- Wash gel with Milli-Q water for 1 min
- Change gel chamber
- Wash gel with Milli-Q water for 1 min
- Develop gel with developer solution
Observe the color and change solution when the developer turns yellow. .Terminate when the staining is sufficient. - Terminate developing with stop solution
Change the solution a couple of times - 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
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