ProTocol A: Digestion of Proteins in Solution

Trypsin (SERVA cat. no. 37283, 37286)

• 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).

Endoproteinase Glu-C (SERVA cat. no. 20984)

• Digestion buffer: 100 mM NH₄HCO_3, 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 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.

Lysyl Endopeptidase^® (Lys-C, SERVA cat. no. 20987)

• Digestion buffer: 50 mM Tris/HCl, pH 8.5
• Reconstitute the lyophilized enzyme in digestion buffer to make a 1 ng/μl solution.
• 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, 37286)

• 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.

Endoproteinase Glu-C (SERVA cat. no. 20984)

• 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.

Lysyl Endopeptidase^® (Lys-C, SERVA cat. no. 20987)

• Digestion buffer: 50 mM Tris/HCl, pH 8.5
• Reconstitute the enzyme in digestion buffer to achieve a final concentration of 10 ng/μl.
• Add 100 μL of the enzyme solution to the dried gel pieces and incubate on ice for 45 min.
• Remove the enzyme solution from the gel pieces with a micro pipettor and replace with 10 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.