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  • Endoproteinase GluC

    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: MALDI-TOF Ms
    Issatchenkia orientalis Cytochrome c subjected to digestion by Endoproteinase GluC for 16 hours, dried and subjected to MALDI-TOF MS.

    Highlights

    • Ideal for proteome analysis
    • Free of contaminating proteases. Produced from a protease-deficient Bacillus subtilis strain
    • Recombinant enzyme

    Product Source

    Staphylococcus aureus Protease V8 gene cloned and expressed in Bacillus subtilis

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    GluC Reaction Buffer-202X

    Properties and Usage

    Reaction Conditions

    1X GluC Reaction Buffer
    Incubate at 37°C

    1X GluC Reaction Buffer:
    50 mM Tris-HCl
    0.5 mM Glu-Glu
    pH 8 @ 25°C

    Usage Concentration

    100 ng/μl

    Storage Temperature

    -20°C

    Molecular Weight

    Theoretical: 29849 daltons

    Notes

    1. Storage conditions: Supplied freeze-dried from Tris-HCl and sodium chloride buffer.
    2. Reconstitution: Endoproteinase GluC should be reconstituted by the addition of 50-500 µl of high purity water. Finger flick the volume of water in the tube to fully resuspend the enzyme. Rapid autolysis is a function of enzyme concentration; any sample reconstituted in a small volume should be used immediately. To get the most use out the enzyme, resuspend the enzyme in 500 ul H2O and aliquot 50 ul each in 10 tubes. Freeze the tubes that are not being used immediately at -20°C for up to two weeks or less. Storage at - 80°C will prolong enzyme stability approximately 2-4 additional weeks.
    3. Repeated freeze/thawing is not recommended.
    4. GluC may also be used in 20-50mM phosphate or ammonium bicarbonate buffer. For optimal activity, supplement reactions with Glu-Glu dipeptide. Glutamic acid residues are highly favored in Tris, phosphate and ammonium bicarbonate buffers.
    5. GluC is purified with a His-tag at its C-terminal of the protein. The predominant species has four histidine residues at the C-terminus with the remaining species showing a distribution from one to six His tags.

    References

    1. Drapeau, G.R., Boily, Y. and Houmard, J. Purification and properties of an extracellular protease of Staphylococcus aureus. J. Biol. Chem.. 247
    2. Benner, J.S., Martin, D. and Schampel, A.K. unpublished results.
    3. Birktoft, J.J. and Breddam, K. Proteolytic Enzymes: Serine and Cysteine Peptidases. A.J. Barrett(Ed.), Glutamyl endopeptidases. Methods Enzymol.. 244, 114-126. San Diego: Academic Press.
    4. Breddam, K. and Meldal, M. Substrate preference of glutamic-acid-specific endopeptidases assessed by synthetic peptide substrates based on intramolecular fluorescence quenching. Eur. J. Biochem.. 206

    FAQs

    1. What is the stability/compatibility of Endoproteinase GluC with regards to presence of DTT, urea, concentrations of methanol, acetonitrile, nitrosoguanidine, thiourea and glycerol?
    2. How does one carry out limited proteolysis experiments with Trypsin and Endoproteinase Glu-C? Which chemicals can be used to quench the reactions at given time points to monitor proteolysis progress via SDS- PAGE?

    Protocols

    1. Typical GluC Digest Reaction Conditions (P8100)
    2. Typical Trypsin and GluC Co-Digest Reaction Conditions (P8100)
    3. Typical GluC In-Gel Digest Reaction Conditions (P8100)

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.

    Selection Charts

    Interactive Tools

    Quality Control

    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.

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page. Further information regarding NEB product quality can be found here.
    • Functional Test (Cytochrome C Digestion):
      The endoproteinase is tested for its ability to digest Issatchenkia orientalis Cytochrome C as determined by MALDI-TOF MS analysis.
    • Specific Activity (Fluorometric):
      The endoproteinase is tested in a reaction with labeled peptide and the initial rate of digestion is determined by measurement of the increase in fluorescence

    Specifications

    The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

    Safety Data Sheet

    The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.