Download TRYPSIN / LYS

LYS-C + TRYPSIN DIGESTION
TCA-precipitated Proteins
 Add 30µl of 100mM Tris-HCl, pH 8.5, 8M Urea (made fresh). Vortex
 Bring solution to 5mM TCEP with 0.1M stock
@ RT, 30 min
 Bring solution to 10mM CAM with 0.5M stock
@ RT, 30min, in dark
 Add 1µl Endoproteinase Lys-C @ 1µg/µl
@ 37°C for >6 hours
 Dilute to 2M Urea with 100mM Tris-HCl, pH 8.5
 Add CaCl2 to 2mM (stock at 500mM)
 Add 5µl Trypsin at 0.1µg/µl
@ 37°C O/N
 Add 90% Formic acid to 5%
Sample

Tris-Urea

TCEP

CAM

Lys-C

Tris

CaCl2

Ti

Formic
30µl
1.5µl
0.6µl
1µl
90µl
0.48µl
5µl
7µl
Lys-C
EC 3.4.21.50
Inhibitors: DFP, TLCK, aprotinin, and leupeptin
Molecular weight: Mr = 33 kD (reduced), Mr = 30 kD (non-reduced)
Optimum pH: 8.5 - 8.8
Specificity: Serine protease that specifically hydrolyzes amide, ester, and peptide bonds at the carboxylic side of
Lys.
Stability: stable in 4 M urea
Trypsin
EC 3.4.21.4
Inhibitors: TLCK, DFP, PMSF, leupeptin, soybean trypsin inhibitor, trypsin inhibitor from hen egg, aprotinin, α2macroglobulin, α1-antitrypsin, APMSF, and antipain
Specificity: Serine endopeptidase that specifically hydrolyzes proteins and peptides at the carboxy side of the basic
amino acids Arg and Lys. Amide and ester bonds of Arg and Lys are also cleaved.
Optimum pH: 8.0
Description Trypsin specifically hydrolyzes peptide bonds at the carboxylic sides of lysine and
arginine residues. Unmodified trypsin is subject to autolysis, generating fragments
that can interfere with protein sequencing, HPLC or mass spectrometry analysis of
the peptides. In addition, autolysis can result in the generation of pseudotrypsin,
which has been shown to exhibit an additional chymotrypsin-like specificity (1).
Promega Trypsin has been modified by reductive methylation, rendering it extremely
resistant to autolytic digestion (2). In functional stability tests, modified trypsin
retains at least two times as much activity as unmodified trypsin after a 3-hour
incubation at 37°C. The sequencing grade of modified trypsin has been further
improved by TPCK treatment followed by affinity purification yielding a highly active
and stable molecule. Sequencing Grade Modified Trypsin is provided in convenient
20μg aliquots with a stability-optimized resuspension buffer. A protease:protein ratio
of 1:100 to 1:20 (w/w) is recommended.
Recommended Reaction Buffer: 50mM NH4HCO3 (pH 7.8).
1