Protocols

Gel staining protocols
Many staining methods are compatible with subsequent tryptic digestion and mass spectrometric analysis. However, it is important that the proteins are not covalently modified or irreversibly fixed in the gel during staining. We prefer that you stain the gels by Coomassie blue stain (Bio-Safe) from Bio-Rad, Cat# 161-0786. If you choose to use silver staining, be careful to follow the suggested protocol below. Otherwise, it may not be possible to obtain any mass spectrometry data.

Coomassie Blue staining

  1. Wash 3 times - 5 min washes with ddH2O
  2. Fix the gel with 40% methanol, 10% acetic acid, and 50% H2O for 60 min
  3. Rinse the gel with ddH2O for 3 times
  4. Wash with 50% ethanol, 50% H2O overnight to decrease the gel background at 4°C
  5. Rinse with ddH2O 3 times
  6. Stain the gel in Bio-Safe™ coomassie stain solution which is enough to cover the gel
  7. Gently shake for 1 hour
  8. Rinse with ddH2O for 3 times
  9. Wash with ddH2O overnight
  10. Wrap the gel with clean Sara wrap. Acquiring gel image can be done with a scanner. Never touch your gel with bare skin.
  11. Store the gel in 1 to 2% acetic acid

SYPRO Ruby staining

  1. Use plastic tray as staining container
  2. Rinse the gel 3 times with ddH2O
  3. Fix the gel with 10% methanol, 7 % acetic acid, and 83% H2O for 60 min
  4. Stain the gel in SYPRO Ruby solution which is at least 10 times the volume of the gel
  5. Gently shake for at least 3 hour for maximal sensitivity or overnight
  6. Wash the gel with 10% methanol, 7 % acetic acid, and 83% H2O for 30-60 minto decrease background fluorescence
  7. Wash the gel 3 times - 5 min with ddH2O
  8. Acquire gel image under a blue light transilluminator (image can be seen by hand-held UV source)
  9. Store the gel in 1-2% acetic acid

Note that use SYPRO Ruby displays difficult to stain glycoproteins and lipoproteins

Silver staining (Adapted from Blum et al, Electrophoresis, 8, pp93-99, 1987 and Schevchenko, A. et al. (1996) Anal. Chem. 68, 850-858)

  1. Fix the gel in 50% ethanol, 5% acetic acid for 1hr.
  2. Wash the gel in 50% ethanol overnight.
  3. Wash gel in Milli Q H2O for 3 times - 10 min
  4. Sensitize gel in 0.02% Na2S2O3.5H2O for 1 min.
  5. Wash gel in chilled H2O, 3 times - 30 secs
  6. Incubate gel in cold 0.1% AgNO3 for 20 mins. at 4°C
  7. Wash the gel in H2O 3 times - 20 secs
  8. Transfer the gel to a clean container
  9. Wash the gel in H2O for 1 min.
  10. Develop the gel in 0.05% Formalin, 3% Na2CO3 (To make 500ml of 0.05% formalin needs 0.25 ml of 37% formaldehyde)
  11. Change the developing solution when the developer turns yellow.
  12. Stop the staining with 5% acetic acid
  13. Wash the gel 3 times - 10 mins with ddH2O
  14. Store the gel at 4°C in 1% HAc

Protein precipitation protocols

Chloroform-Methanol Precipitation

  1. To sample of starting volume of 100 ml
  2. Add 400 ml methanol
  3. Vortex well
  4. Add 100 ml chloroform
  5. Vortex
  6. Add 300 ml H2O
  7. Vortex
  8. Spin 1 min @ 14,000 x g
  9. Remove top aqueous layer (protein is between layers)
  10. Add 400 ml methanol
  11. Vortex
  12. Spin 2 min @ 14,000 x g
  13. Remove as much methanol as possible without disturbing pellet
  14. Speed-vac to dryness
  15. Bring up in 2X sample buffer for SDS-PAGE

TCA Precipitation

  1. Dilute protein to 500 µL with water in eppendorf tube
  2. Add:
    • 50µL of DOC
    • 25 µL of TX 100
    • 80 µL of TCA (13%TCA)
  3. At least 2 hrs on ice
  4. Spin in a bench-top microfuge at 4°C for 30 min at Max. speed (15,000 rpm).
  5. There will be a flaky pellet up the side of the tube
  6. Dump sup, let the tube stand on end on a kimwipe to get most of the liquid out. Don't sweat it if a little is left in the bottom
  7. Add 500µL of ethanol/ether( cold aceton) . Bath sonicate to thoroughly disrupt pellet
  8. On ice for 30 min
  9. Repeat step 4
  10. There will be little to no pellet
  11. Repeat step 6
  12. Let air dry at room temperature
  13. Re-suspend samples in 5 µL running buffer first, then 20 µL sample buffer
  14. Heat at 85°C for 5 min.
  15. Load sample to each well (25µL)

References