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Endoproteinase AspN |
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- Ideal for proteome analysis
- Free of contaminating proteases
- Recombinant enzyme
Description:
Endoproteinase AspN (flavastacin) is a zinc metalloendopeptidase which selectively cleaves peptide bonds N-terminal to aspartic acid residues (1).
Reconstitution:
Endoproteinase AspN should be reconstituted by the addition of 50-500 µl of high purity water. Rapid autolysis is a function of enzyme concentration.
Source:
Chryseobacterium [Flavobacterium] menigosepticum gene cloned and expressed in Chryseobacterium [Flavobacterium] menigosepticum.
Applications:- Digestion of proteins for proteomic analysis by Mass Spectrometry
- Protein and peptide identification
Reagents Supplied:
AspN ReactionBuffer (2 X)
Enzyme Properties
Molecular Weight:
Theoretical: 40,089.9 daltons
Specific Activity:
25 μmol/min/mg
Reaction & Storage Conditions
Reaction Conditions:
1X AspN ReactionBuffer
Incubate at
37°C.
1X AspN ReactionBuffer:
50 mM Tris-HCl
2.5 mM ZnSO4
pH 8.0 @ 37°C
Storage Temperature:
-20°C
Notes
General notes:- Storage Conditions: Supplied in lyophilized form. Can be stored frozen in solution at -20°C for up to 2 weeks. A decrease in activity will occur if stored in solution. Use only freshly reconstituted protease for best results.
- Aspartic acid residues are strongly favored by Endoproteinase AspN in all buffer conditions we have examined (Tris-HCl, ammonium bicarbonate and potassium phosphate) (2).
The specificity of Endoproteinase AspN is based on the three sited references and should be considered provisional at this time.
Endoproteinase AspN contains a O-linked carbohydrate on the protein. The average protein appears as a single band by SDS-PAGE analysis.
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:
Endoproteinase AspN is free of glycerol and detergents which may interfere with Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) or Electrospray Ionization (ESI) Mass Spectrometry (MS), or liquid chromatography (LC) methods.
Endoproteinase AspN Activity:
Measured in three assays:
1. Protein digestion and analysis by MALDI-TOF MS or ESI-TOF MS
2. Peptide digestion and analysis by MALDI-TOF MS or ESI-TOF MS
3. Fluorometric substrate digestion and specific activity determination in digestion buffer: 50 mM Tris-HCl (pH 8.0) and 2.0 mM Zinc Acetate.
Protein Digestion:
Issatchenkia orientalis Cytochrome c (Swiss-Prot: CYC_ISSOR) (Sigma) is subjected to digestion by AspN at a ratio of 20:1 respectively for 16 hours at 37°C in AspN 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 Waters MALDI micro MX MALDI-TOF MS, or ESI-MS using an Agilent ESI-TOF after separation by C18 reverse-phase HPLC.
Peptide Digestion:
ACTH (18–39) peptide is subjected to digestion by AspN at a ratio of 20:1 respectively for 16 hours at 37°C in AspN 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 Waters MALDI micro MX MALDI-TOF MS, or ESI-MS using an Agilent ESI-TOF after separation by C18 reverse-phase HPLC.
Fluorometric Assay:
1 µg (~1 µmol) of Anthranilyl-Ala-Phe-Ala-Phe-Asp-Val-Phe(NO2)-Tyr-Asp peptide (Sigma) was suspended in 150 µl of AspN Reaction Buffer and 1 µg of Endoproteinase AspN was added (4). The initial rate was determined by measurement of the increase in fluorescence (excitation 330 nm and emission 450 nm). The protein concentration is determined by C18 reverse-phase HPLC and integration.
Endoproteinase AspN Protein Sequence
MALDI-TOF MS: Issatchenkia orientalis Cytochrome c subjected to digestion by Endoproteinase AspN for 16 hours, dried and subjected to MALDI-TOF MS.
References
- Tarentino A.L. Flavastacin (2004) In Handbook of Proteolytic Enzymes, 2nd Ed., 631-632, Elsevier, London.
- Tarentino, A.L. et al. (1995) Arch Biochem Biophys, 319, 281-285.
- Grimwood, B.G., Plummer, T.H. and Tarentino, A.L. (1994) Arch Biochem Biophys, 311, 127-132.
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