Protocols

ProTocol A: Digestion of Proteins in Solution

Trypsin (SERVA cat. no. 37283, 37284, 37284)

  • Lyophilized Trypsin is reconstituted in 50 mM acetic acid to a final concentration of 1 µg/µl.
  • For digestion of the target protein, add Trypsin to a final protease:protein ratio of 1:100 to 1:20 (w/w).

Endproteinase Glu-C (SERVA cat. no. 20986)

  • Digestion Buffer: 100 mM NH4HCO3, pH 7.8 (optional: 100 mM Tris/HCl, pH 7.8)
  • Reconstitute the lyophilized enzyme in deionized water to make a 100 ng/μl solution.
  • Prepare a 5 ng/μl enzyme in Digestion Buffer solution by combining 5 μl of the 100 ng/μl enzyme solution with 95 μl Digestion Buffer.
  • The specific volumes and concentration can be adjusted for sample numbers and/or quantity of protein in the sample.
  • A final enzyme to protein ratio of 1:20 to 1:100 is recommended.

ProTocol B: In-gel Protein Digestion

Trypsin (SERVA cat. no. 37283, 37284, 37284)

  • Lyophilized Trypsin is reconstituted in 50 mM acetic acid to a final concentration of 1 µg/µl.
  • Add 25 mM NH4HCO3, pH 8 to get a final Trypsin concentration of 50 µg/ml.
    For example: 25 µl Trypsin solution (1 µg/µl) + 475 µl NH4HCO3, pH 8 (25 µg Trypsin/500 µl = 50 µg Trypsin/ml)
  • For the final use dilute the Trypsin solution 1:2.5 with 25 mM NH4HCO3, pH 8 and use 10 – 20 µl for rehydration/digestion of each gel piece.

Endproteinase Glu-C (SERVA cat. no. 20986)

  • Digestion Buffer: 100 mM NH4HCO3, pH 7.8 (optional: 100 mM Tris/HCl, pH 7.8)
  • Combine 10 μl of the 100 ng/μl reconstituted enzyme with 90 μl of Digestion Buffer to achieve a final concentration of 10 ng/μl. Place on ice until ready to use.
  • Add 50 μL of the 10 ng/μl enzyme solution to the dried gel pieces and incubate on ice for 45 min.
  • Remove the 10 ng/μl enzyme solution from the gel pieces with a micro pipettor and replace with 50 μl of digestion buffer.
  • Incubate overnight at 37 °C.

Peptide Extraction (Recommended to avoid clogging of the LC system)

  • Clear solution of the In-Gel digest by centrifugation and transfer the supernatant into a new vial.
  • Alternative method: Extraction of the peptides, e.g. with acetic acid and actonitrile.

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