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Endoproteinase GluC |
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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. Produced from a protease-deficient Bacillus subtilis strain
- Recombinant enzyme
Description:
Endoproteinase GluC (Staphylococcus aureus Protease V8) is a serine proteinase which selectively cleaves peptide bonds C-terminal to glutamic acid residues (1). Endoproteinase GluC also cleaves at aspartic acid residues at a rate 100-300 times slower than at glutamic acid residues (2,3).
MALDI-TOF MS: Issatchenkia orientalis Cytochrome c subjected to digestion by Endoproteinase GluC for 16 hours, dried and subjected to MALDI-TOF MS.
Source:
Staphylococcus aureus Protease V8 gene cloned and expressed in Bacillus subtilis
Reagents Supplied:
GLuC Reaction Buffer
Enzyme Properties
Molecular Weight:
Theoretical: 29,849 daltons
Specific Activity:
38.3 μmol/min/mg
Reaction & Storage Conditions
Reaction Conditions:
1X GLuC Reaction Buffer
Incubate at
25°C.
1X GLuC Reaction Buffer:
50 mM Tris-HCl
0.5 mM Glu-Glu
pH 8.0 @ 25°C
Storage Temperature:
-20°C
Supplied freeze-dried from a potassium phosphate and sodium chloride buffer.
Notes
General notes:- Glutamic acid residues are strongly favored by Endoproteinase GluC in all buffer conditions we have examined (Tris-HCl, ammonium bicarbonate and potassium phosphate) (2).
- Endoproteinase GluC contains a 6-His-Tag on the C-terminal of the protein. The average protein appears as a single band on SDS-PAGE and a small amount of this protein may contain two extra Ala residues at the N-terminus of the protein (2). This sequence is also available at www.neb.com.
- Store frozen 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.
- Reconstitution: Endoproteinase GluC should be reconstituted by the addition of 50-500 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:
Endoproteinase GluC 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.
Endoproteinase GluC 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) and 0.5 mM Glu-Glu.
Protein Digestion::
Issatchenkia orientalis Cytochrome c (Swiss-Prot: CYC_ISSOR) (Sigma) is subjected to digestion by GluC at a ratio of 20:1 respectively for 16 hours at 25°C in GluC 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::
ACTH (1-17) peptide is subjected to digestion by GluC at a ratio of 20:1 respectively for 16 hours at 25°C in GluC 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::
1 µg (~1 µmol) of Anthranilyl-Ala-Phe-Ala-Phe-Glu-Val-Phe(NO2)-Tyr-Asp peptide (Sigma) was suspended in 150 µl of GluC Reaction Buffer and 1 µg of Endoproteinase GluC 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.
References
- Drapeau, G.R., Boily, Y. and Houmard, J. (1972) Purification and properties of an extracellular protease of Staphylococcus aureus. J. Biol. Chem., 247, 6720-6726.
- Benner, J.S., Martin, D. and Schampel, A.K., unpublished observations.
- Birktoft, J.J. and Breddam, K. (1994) In Proteolytic Enzymes: Serine and Cysteine Peptidases. A.J. Barrett (Eds.), Glutamyl endopeptidases. Methods Enzymol., 244, pp. 114-126. San Diego: Academic Press.
- Breddam, K. and Meldal, M. (1992) Substrate preference of glutamic-acid-specific endopeptidases assessed by synthetic peptide substrates based on intramolecular fluorescence quenching. Eur. J. Biochem., 206, 103-107.
Reagents Sold Separately
GLuC Reaction Buffer
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