Preparing SDS-PAGE Gels for Protein Identification by Mass Spectrometry
Before running the gel
Polymerize acrylamide gels overnight to eliminate cross-linking of unpolymerized acrylamide to proteins during electrophoresis. Always wear gloves (powderless, rinsed with water and ethanol before use) to eliminate contamination by keratins, etc. Optimum thickness for gels is 1 mm. Also, it is desirable to achieve as high a ratio of protein to gel volume as possible, i.e. to use the narrowest gel lane width possible.
After running the gel
Use CLEAN dishes for staining the gels (no BSA from Western blots, etc., unless you want us to identify these proteins instead of your interesting proteins), and freshly made staining solutions. Please be sure to follow one of the staining protocols shown below because many staining protocols, though perhaps slightly more convenient or sensitive than one of these, may well render your proteins impervious to mass spectrometric analysis. That said, the general rule is that most Coomassie stains are OK, but in all cases chemical crosslinkers and strong oxidizing or reducing agents (included in many silver staining protocols) should be avoided. Recently several commercial silver staining kits that give good staining results and also are compatible with downstream mass spectrometry analysis have been developed. One such kit we have tried successfully is made by Invitrogen (SilverQuestTM Silver Staining Kit, catalog # LC6070). After staining, gels may be stored in 1% acetic acid. Ideally the whole gel is sent to us if it is possible to clearly designate which bands we should cut out and identify. It is also preferable if you allow us to acquire images of the gel before excising bands of interest. If you acquire an image (i.e. photo or scan), take special care not to allow your gel to contact any contaminated surfaces during the process of acquiring an image of your gel- again, it is preferable to let us do this for you. If you decide to excise the bands in your lab, be sure to use extremely clean surfaces and new razor blades or scalpels if possible. Ideally this should be done in a laminar flow hood (tissue culture type) to minimize the possibility of any dust, hair, flakes of skin, or other forms of dirt. Even trace amounts of such contaminants usually contain keratins in much larger amounts than the proteins present in your gel bands of interest and therefore overwhelm any attempt to analyze these bands. Once cut, gel bands can be stored frozen in water or 1% acetic acid in clean, sealed sample tubes. It is best to ship gel bands to us frozen if possible.
Gel Staining Procedures
Coomassie Brilliant Blue Staining
- Fix gel in 100 ml 46 % MeOH, 7 % HAc for 1 hour.
- Stain gel in 100 ml 46 % MeOH, 7 % HAc, 0.1% Coomassie Brilliant BlueæR-250 (filter this before use) for 1 hour. Destain gel in100 ml 5% MeOH, 7.5% HAc for 24 hours. Replace if needed.
- Store the gel in 1 to 2% HAc.
(Schevchenko, A. et al. (1996) Anal. Chem .68 , 850-858)
- Fix the gel in 50% MeOH, 5% HAc for 20 mins.
- Wash the gel in 50% MeOH for 10 mins.
- Wash gel in Milli Q H2O for at least 2 hrs- overnight washing is better because it will reduce the staining background
- Sensitize gel in 0.02% Na2S2O3for 1 min.
- Wash gel twice in H2O for 1 min. each time
- Incubate gel in cold 0.1% AgNO3 for 20 mins. at 4ÅC
- Wash the gel in H2O for 1 min.
- Transfer the gel to a clean container
- Wash the gel in H2O for 1 min.
- Develop the gel in 0.04% Formalin (35% formaldehyde), 2% Na2CO3.
- Change the developing solution when the developer turns yellow.
- Stop the staining with 5% HAc
- Change the 5% HAc solution at least twice
- Store the gel at 4Å C in 1% HAc