Preparing and Running 1D or 2D SDS-PAGE Gels for analysis by MALDI-TOF/TOF
In-gel tryptic digestion with mass spectrometric analysis by MALDI-TOF-TOF and subsequent database search is the most common method used for protein identification. However, the identification becomes needlessly laborious when contaminant peaks dominate the mass spectra.
The most common contaminant in SDS-PAGE gels is keratin. During mass spectrometric analysis the keratin-derived peptide signals are often stronger than those of the samples. In most cases where the concentration of the protein(s) of interest approaches the limit of detection, keratin contamination becomes especially critical as this can prevent the identification of the sought after protein(s). The chemicals used for the preparation of gels can also affect the separation during SDS-PAGE hence it is necessary to ensure that they are free from contamination and are freshly prepared when necessary. The following are some helpful tips when working with SDS-PAGE gels.
- Always wear gloves when handling gels.
-
If you are pouring your own gels,
- Wash spacers and glass plates thoroughly with deionized water and 100% methanol. Dry under the hood prior to pouring the acrylamide solution. If necessary, wash the plates in liquid Alconox or RBS-35 (Pierce).
- Always check your TEMED and persulfate solution as these can affect the polymerization of your gel. If you see wave-like formations in your gels, these are indications that it did not polymerize well.
- If you added water-saturated isobutanol on the top of the gel, do not leave the isobutanol overlay for more than 2 hours
- Completely remove the overlay by washing the top of the polymerized gel with deionized water and then finally with gel storage buffer
- Store the gel in gel storage buffer at 4oC at least overnight before using it to allow for complete polymerization.
- Extensive handling of gel occurs during the fixing/staining process, especially when silver staining is used as this requires changing solutions several times. Make sure that you use clean gloves and to change them as often as necessary.
- When preparing the bands for in-gel digestion,
- Wash all glassware/ materials with deionized water and 100% methanol. Air dry in a clean hood.
- Wash the gel thoroughly with deionized water.
- Cut the gel bands in a clean hood. Use a different blade for each band or wash with water and methanol before using it to cut the next band.