Gel electrophoresis:
Common mistakes in electophoresis:

Gel staining - Colloidal coomassie staining
Colloidal blue staining kit from Invitrogen

Laboratory protocols:
Please visit

References:

• Neuhoff V, Stamm R, Pardowitz I, Arold N, Ehrhardt W, Taube D, Electrophoresis. 1990, 11, 101-117
• Neuhoff V, Arold N, Taube D, Ehrhardt W, Electrophoresis 1988, 9, 255-262

Gel staining - Silver staining
Protocols employing glutardialdehyde are not compatible with mass spectrometry! Commercial kits: see SilverQuest kit and Silver stain MS kit

Protocol:

1. Fix gel in 50% MeOH, 12% AcOH, 0.05% formalin for 2 hrs
2. Wash gel 3x in 35% EtOH for 20 mins each
3. Wash gel 2x 10 mins in H₂O
4. Sensitize gel in 0.02% Na₂S₂O₃ for 2 mins
5. Wash gel 3x in H₂O for 5 mins each
6. Stain gel with 0.2% AgNO₃, 0.076% formalin for 20 mins (30 mins at 4° to 6° C leads to lower background)
7. Wash gel in H₂O for 2x 1 min each
8. Develop gel in 6% Na₂CO₃, 0.05% formalin, 0.0004% Na₂S₂O₃
9. Wash once with H₂O
10. Stop staining with 50% MeOH, 12% AcOH (at least 5 mins)
11. Wash once with H₂O
12. Optional: block any residual formalin with 5% glycine (5 mins) and wash once with H₂O
13. Store gel in 1% AcOH at 4° to 6° C

References:

• Mortz E, Krogh TN, Vorum H, Gorg A, Proteomics. 2001, 1, 1359-63

Keratin free environment
Generally, in a standard laboratory setup it is not possible to work completely keratin free, but the amount of contamination can be substantially reduced by following a few guidelines:

• Buffers and solutions should be free of dust and any other particulate material (optional: filter solutions).
• Exclusively use Milli Q grade water.
• Never bring the sample into contact with anything that may have touched fingers or had dust fall on it.
• Work with nitril gloves (latex gloves can still contain proteins).
• Use clean plastic wear, pipette tips and plastic trays.

see also this PDF from thermo.com

Gel systems: NuPAGE from Invitrogen
Bis-Tris based gel system
High quality acrylamide/polyacrylamide stock solutions can be purchased from National Diagnostics
References:

• Wiltfang J, Arold N, Neuhoff V, Electrophoresis 1991, 12, 352-366
  Katayama, H et al., Rapid Commun Mass Spectrom. 2004, 18, 2388-2394

Sample concentration
Millipore or Vivascience 3K centrifugal devices: can be washed out with SDS sample buffer after concentration to minimize sample loss.

1. Add glycogen (Roche, molecular biology grade) to a final concentration of 20 µg to ml
2. Add 3 volumes of 96% ethanol (analytical grade). For samples containing denaturants/chaotropic salts (urea, guanidinium hydrochloride, thiourea, rhodanide) add 5 volumes of ethanol
3. Add 0.04 volumes of 2.5 M sodium acetate, pH 5.0 (final concentration 0.1 M)
4. Mix and incubate at room temperature for at least 3 hrs
5. Centrifuge at > 13 000xg for 15 mins at room temperature and aspirate supernatant (optional wash of pellet with 70% ethanol, not really necessary). Do not dry the pellet
6. Dissolve in buffer of choice (SDS sample buffer)

Disclaimer: samples with NaCl concentrations > 400 mM should be diluted to avoid sample loss due to insolubility of precipitate; evaluate performance with a test sample

Commercial precipitation kits: We recommend the GE Healthcare Biosciences
2D gel clean-up kit (#80-6484-51)