Protocols

Gel staining

The use of high quality chemicals and freshly prepared solutions using pure deionised water is strongly recommended. Thin gels (less than 1mm) should be preferred if possible to maximize protein recovery during the extraction step. When dealing with low molecular weight proteins (< 25kD), high acrylamide gel percentages are recommended. 

Classical Coomassie Blue:

  • Wash the gel at the end of migration in water for a couple of minutes (optional if dealing with very small proteins or peptides). 
  • Stain the gel for 2 hours or overnight in 0.1% Coomassie Blue R-250, 10% acetic acid, 50% Methanol. Change solution 2 or 3 times during the staining process to remove excess of SDS.
  • De stain the gel in 10% acetic acid, 50% Methanol. Change destaining solution several times until clear background is obtained.
  • Gel can be stored in 10% ethanol at 4°C.

Alternatively an increased staining sensitivity can be reached when using Colloidal Brilliant Blue G-250
(Electrophoresis. 2004 May;25(9):1327-33).

Silver staining compatible with mass spectrometry:

Several protocols are available nowadays; the EPFL-PCF selected procedure is described below (Proteomics. 2001, 1, 1359-1363).

  • Fix the gel for 1 hour in 40% Ethanol, 10% acetic acid.
  • Wash the gel 2 x 20min. in 30% Ethanol.
  • Wash the gel for 20min. in pure deionised water.
  • Sensitize the gel for 1min. in 0.02% Na2S2O3 .
  • Wash the gel 3 x 20sec. in pure deionised water.
  • Incubate the gel for 20min. in cold 0.1% AgNO3 at 4°C.
  • Wash the gel 3 x 20sec. in pure deionised water
  • Change gel recipient.
  • Wash the gel for an additional minute in pure deionised water.
  • Develop the gel in 3% Na2CO3 , 0.05% formalin. Follow the colour change of the developer solution and change it when it turns yellowish. Terminate when you estimate the staining is sufficient.
  • Wash the gel for 20sec. in pure deionised water
  • Finalize the staining in 5% acetic acid.
  • Wash the gel 3 x 10mins. in pure deionised water. 
  • Store the gel in 1% acetic acid at 4°C.

In-Gel digestion

High quality chemicals and pure deionised water should be used. Major precautions should always be taken during gel handling processes to avoid keratins contaminations which can severely interfere with the MS detection of your analysed sample.

Wash all experiment devices (gel cutting surface, razor blades, solution bottles, instruments such as Speed Vac and centrifuge chambers, internal and external sample tube surfaces) with an acidified mixture of aqueous and organic solvent. Wear powder free gloves and avoid touching organic solvent with them to minimise polymer contamination. Tie off your hair and work under laminar flow hood. Cut the gel under a pure deionised water film.

     Ammonium bicarbonate: AB (Fluka: 09830)
     Iodoacetamide: IAA (Fluka: I 1149)
     Dithioerythritol: DTE (VWR/Merck: 1.24511)
     Acetonitrile: CAN (Fischer Scientific: A/0626/17)
     Formic acid: FA (VWR/Merck: 1.00264)
     Trypsin: (Catalys AG: V 5280)

Gel washing step:

  • Cut the gel band or spot, slice it and collect pieces on a clean PCR or a 500 µl tube.
  • Spin down and remove excess of water using a gel loader tip. 
  • Add 150 µl of 50% Ethanol, 50% 50 mM AB (pH 8.4), make sure the gel is well soaked and incubate 20 min. at room temperature.
  • Spin down, remove liquid and repeat previous step once.
  • Spin down, remove liquid and dry the gel in Speed Vac (around 20 min).

Reduction and alkylation (if necesary):

  • Cover the gel piece (25-30 µl) with freshly prepared 10 mM DTE, 50 mM AB (pH 8.4). Vortex briefly, spin down and incubate 1h00 at 37°C protecting from light.
  • Remove DTE solution and add 25-30 µl of freshly prepared 55 mM IAA, 50 mM AB (pH 8.4). Vortex briefly, spin down and incubate 45min. at 37°C protecting from light. 
  • Remove IAA solution and wash 2-3 times with 50mM AB (pH 8.4). Dehydrate the gel by adding 100µl of 50% Ethanol, 50% 50 mM AB (pH 8.4) and incubate at room temperature for 20 min.
  • Spin down, remove the liquid and Speed Vac to complete gel dryness (around 30 min). Store sample tubes on ice.

Tryptic digestion:

  • Cover the gel piece (25-30 µl) with a trypsin solution made of 12.5ng/µl in 50 mM AB (pH 8.4), 10mM Calcium Chloride. Vortex shortly, spin down and incubate 10mins. on ice.
  • Check liquid volume and add enough 50 mM AB (pH 8.4) solution to cover the gel piece. Vortex shortly, spin down and incubate again for 10mins. on ice.
  • Check again liquid volume, add 50 mM AB (pH 8.4) if necessary, seal PCR tube and incubate at 37°C over night.

Peptides extraction:

  • If liquid volume is not covering the gel, add 50 mM AB (pH 8.4) and incubate for an additional hour.
  • Spin down and collect supernatant into a clean PCR tube (keep same pipet tip for next extraction steps).
  • Add 10 µl of 5% FA, 50% Ethanol and vortex for 20 min. Spin down, collect supernatant and pool it with previous one (0.5µl can be here directly spotted on MALDI plate for TOF analysis).
  • Add 30 µl of 5% FA, 70% Ethanol and vortex for 20 min. Spin down, collect supernatant and pool it with previous ones.
  • Speed vac pooled samples to complete dryness.
  • Resuspend in 50 µl of 100% ACN and Speed vac again to complete dryness to concentrate the sample
  • Repeat last step once. 
  • Resuspend dry sample in 20 µl of 2% ACN, 20% FA and inject on LC-MS.