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Endoproteinase AspN |
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Prices are in US dollars and valid only for US orders.
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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:
Purified from Flavobacterium menigosepticum.
Applications:- Digestion of proteins for proteomic analysis by Mass Spectrometry
- Protein and peptide identification
Reagents Supplied:
Endoproteinase AspN Reaction Buffer (2X)
Enzyme Properties
Molecular Weight:
Theoretical: 40,089.9 daltons
Specific Activity:
25 μmol/min/mg
Reaction & Storage Conditions
Reaction Conditions:
1X Endoproteinase AspN Reaction Buffer
Incubate at
37°C.
1X Endoproteinase AspN Reaction Buffer:
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).
Endoproteinase AspN is recommended for cleavage of peptides only. The cleavage rate of protein is much slower.
Endoproteinase AspN contains a O-linked carbohydrate on the protein. The average protein appears as a single band by SDS-PAGE analysis.
FAQs
- Is your Endoproteinase AspN the same as the Endoproteinase AspN sold by other companies?
- The Endoproteinase AspN is not fully dissolving when I reconstitute it in water. How do I get it completely into solution?
- Which residues does Endoproteinase AspN cut?
- I can’t get Endoproteinase AspN to digest my protein.
- Do you have a protocol or pointers for digesting proteins in-gel with endo- proteinase Asp-N followed by Endoproteinase Glu-C? I'm following the protocol for each but would like to streamline the combined digest.
- How pure is Endoproteinase AspN? Has it been checked for nuclease activity?
- Tris will interfere with my reactions downstream. Does your Endoproteinase AspN, when reconstituted in H2O, contain Tris-HCl?
- What is the Endoproteinase AspN sequence in text format?
- Can Endoproteinase AspN be used with a volatile buffer?
- How stable is this enzyme? We dissolved our enzyme in water approx 2 weeks ago and the literature indicates that activity will start to drop after this length of storage time(and longer).
- I digested BSA with Endoproteinase AspN following standard conditions and examined the released fragments with Mass-spec. I did not see many peaks that matched the predicted fragment but saw a lot of unpredicted bands, perhaps indicating non-specific enzyme activity.
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.
Reagents Sold Separately
Endoproteinase AspN Reaction Buffer
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New
England Biolabs, Inc. is an ISO 9001 and ISO
14001 Certified Company.
NEB certifies that it is a small business in accordance with the US Small Business Administration and 13 CFR 121.201 |
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