PROTOCOLS

Gel electrophoresis and subsequent staining is a routine procedure prior to protein identification. Most often SDS-Gels are used. If you don’t use commercial gels, it is of utmost importance that gel-hardening is completed over night. 

Staining

Not all coomassie stains are compatible with mass spectrometry. We strongly recommend that you mix your own coomassie staining solution directly prior to use. Alternatively, visualization can be performed with silver staining when protein concentration is very low.

Cutting of gel bands

Please take precautions to prevent contamination (keratins, etc) when cutting gel bands and consider the following recommendations:

  • Wear gloves during all working steps
  • Place the gel on a clean glass plate on a light box
  • Keeping the gel moist will ease cutting and transfer
  • Use a new scalpel knife
  • Cut bands tightly; including unstained gel will only increase background interference in MS analysis
  • Cut individual bands in cubes of ~1 x 1mm
  • Transfer the gel pieces to an eppendorf tube (1 band per tube)
  • After cutting, gel bands can be kept for months at -20 before MS analysis without adverse effect

Alternatively, the whole gel can be transferred to the PCF.

Gel slices are submitted to tryptic digestion and peptides are extracted prior to LC/MS. For MALDI-TOF-MS the samples have to be desalted using a ZipTip protocol.