 |
|
 |
|
|
| Home >
Products >
Protein Tools >
Proteome Analysis >
Modified Trypsin (TPCK-treated) |
 | | | | Modified Trypsin (TPCK-treated) | | | |
 |
|
Prices are in US dollars and valid only for US orders.
|
- Acetylated to prevent autolysis
- Produces no additional species upon extended digestion
- TPCK treatment eliminates chymotryptic activity
- Ideal for proteome analysis
Description:
Modified Trypsin (TPCK-treated) is a serine endopeptidase. It selectively cleaves peptide bonds C-terminal to lysine and arginine residues (1). Modified Trypsin is treated with L-(tosylamido-2-phenyl) ethyl chloromethyl ketone (TPCK) to inactivate any remaining chymotryptic activity. It is modified by acetylation of the e-amino groups of lysine residues to prevent autolysis. Modified Trypsin cleaves at Lys-Pro and Arg-Pro bonds at a much slower rate than other amino acid residues (2).
Source:
Isolated from bovine (Bos taurus) pancreas
Reagents Supplied:
Modified Trypsin Reaction Buffer
Enzyme Properties
Molecular Weight:
Theoretical: 23,675 daltons
Specific Activity:
2.1 μmol/min/mg
Reaction & Storage Conditions
Reaction Conditions:
1X Modified Trypsin Reaction Buffer
Incubate at
25°C.
1X Modified Trypsin Reaction Buffer:
50 mM Tris-HCl
20 mM CaCl2
pH 8.0 @ 25°C
Storage Temperature:
-20°C
Supplied freeze-dried from a sodium acetate and calcium chloride buffer.
Notes
General notes:- Modified Trypsin is acetylated on multiple lysine residues. This protein appears as a single band on SDS-PAGE. This sequence is also available at www.neb.com.
- Store frozen at -20°C for up to one week. A decrease in activity will occur if stored in solution. Use only freshly reconstituted protease for best results.
- Reconstitution: Modified Trypsin should be reconstituted by the addition of 20-200 µl of high purity water. Rapid autolysis is a function of enzyme concentration.
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Quality Assurance Statement:
Modified Trypsin is free of glycerol and detergents which may interfere with Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) Mass Spectrometry (MS) or liquid chromatography (LC) methods.
Modified Trypsin Activity::
Measured in three assays:
1. Protein digestion and analysis by MALDI-TOF MS
2. Peptide digestion and analysis by MALDI-TOF MS
3. Fluorometric substrate digestion and specific activity determination in digestion buffer: 50 mM Tris-HCl (pH 8.0).
Protein Digestion::
Issatchenkia orientalis Cytochrome c (Swiss-Prot: CYC_ISSOR) (Sigma) is subjected to digestion by Modified Trypsin at a ratio of 20:1 respectively for 16 hours at 25°C in Modified Trypsin Reaction Buffer. 1 µl of the above reaction (50 ng) was mixed with 1 µl of α-cyano-4-hydroxycinnamic acid matrix solution, air-dried and subjected to MALDI-TOF MS analysis on an ABI Voyager DE MALDI-TOF MS.
Peptide Digestion::
A modified Human calcitonin peptide is subjected to digestion by Modified Trypsin at a ratio of 20:1 respectively for 16 hours at 25°C in Modified Trypsin Reaction Buffer. 1 µl of the above reaction (10 ng) was mixed with 1 µl of α-cyano-4-hydroxycinnamic acid matrix solution, air-dried and subjected to MALDI-TOF MS analysis on an ABI Voyager DE MALDI-TOF MS.
Fluorometric Assay::
250 ng (~1 µmol) of Ala-Phe-Lys 7-amidomethyl coumarin peptide was suspended in 150 µl of Modified Trypsin Reaction Buffer and 1 µg of Modified Trypsin was added. The initial rate was determined by measurement of the increase in fluorescence (excitation 365 nm and emission 440 nm). The protein concentration is determined by C18 reverse-phase LC and integration.
References
- Northrop, J.H. and Kunitz, M. (1931) Isolation of protein crystals possessing tryptic activity. Science, 73, 262-263.
- Perona, J.J. and Craik, C.S. (1995) Structural basis of substrate specificity in the serine proteases. Protein Sci., 4, 337-360.
Reagents Sold Separately
Modified Trypsin Reaction Buffer
|
|
|
 |
|
|
