Proteins in polyacrylamide gel

  • Samples for protein identification by proteolytic digestion and LC-MS/MS are commonly submitted as Coomassie stained or silver stained bands or spots in polyacrylamide gels. Optimized staining protocols for samples for MS analysis can be found here. Unstained gels will not be analyzed!
  • The gels should be handled as little as possible, to minimize contamination by dust and keratin.
  • The spot or band of interest should be excised precisely, excluding all of the surrounding blank gel.
  • Cut the excised gel into small pieces (1-2 mm square) and place it in a clean eppendorf vial without addition of buffers/solvents/liquids.
  • If a specific protein should be analyzed in detail (e.g. identification of posttranslational modifications), the protein should be isolated (e.g. by affinity purification) before MS analysis.

Proteins in solution

  • If protein samples are submitted for in-solution digest, the concentration should be at least 20pmol in 1µl (e.g. 0.2µg/µl for a protein with a MW of 10kD). The minimum volume should be 25µl.