• My NEB
  • Print
  • PDF
  • 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: 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-201X

    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

    Specific Activity

    38.3 μmol/min/mg/

    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.
    • 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

    Notes

    1. 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.
    2. Glutamic acid residues are strongly favored by Endoproteinase GluC in all buffer conditions we have examined (Tris-HCl, ammonium bicarbonate and potassium phosphate) (2).
    3. 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.
    4. Storage Conditions: Supplied freeze-dried from a Tris-HCl and sodium chloride buffer.
      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.

    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

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    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.
    1. Is there a compatible buffer for digesting with Trypsin (P8101S) and Endoproteinase GluC (P8100S) together?
    2. How pure is Endoproteinase GluC? Is it sequencing grade?
    3. Is the 50ug of Endoproteinase GluC all in one vial or it is divided into several smaller portions into several vials? How can it be kept stable since we probably won’t be able to use it all in a short period of time.
    4. Why is there 0.5 mM Glu-Glu in the buffer and what is it?
    5. Is Endoproteinase GluC’s activity affected by 0.05% Tween 20?
    6. We have been using your Endoproteinase GluC as a Histidine-tagged protein marker for western blots. The molecular weight listed on your website for this product is 29849 Daltons. Does that include the 6-his added as the tag?
    7. Has anyone ever compared the Roche product along side NEB's? Are they a close to exact equivalent?
    8. What is the stability/compatibility of Endoproteinase GluC with regards to presence of DTT, urea, concentrations of methanol, acetonitrile, nitrosoguanidine and thiourea?
    9. Can Endoproteinase GluC be used to digest substrate in buffer 50 mM Tris, 5% glycerol?
    10. What is the unit definition and/or specific activity of Endoproteinase GluC enzyme?
    11. I would like to perform an in-gel digest using Endoproteinase GluC for subsequent mass spectrometry analysis of an unknown protein. Can you please provide me with a protocol or direct me to an appropriate reference?
    12. 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?
    13. Does Endoproteinase GluC work in phosphate buffer? Ammonium bicarbonate buffer?
    14. Is Endoproteinase GluC active at lower concentrations of digestion buffer?

    Selection Tools