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  • Trypsin-ultra™, Mass Spectrometry Grade

    Description

    Trypsin-ultra™, Mass Spectrometry Grade is a serine endopeptidase. It selectively cleaves peptide bonds C-terminal to lysine and arginine residues (1). Trypsin-ultra is treated with L-(tosylamido-2-phenyl) ethyl chloromethyl ketone (TPCK) to inactivate any remaining chymotryptic activity. It is modified by acetylation of the ε-amino groups of lysine residues to prevent autolysis. Trypsin-ultra (TPCKtreated) cleaves at Lys-Pro and Arg-Pro bonds at a much slower rate than other amino acid residues (2).

    P8101

    MALDI-TOF MS: Issatchenkia orientalis Cytochrome c subjected to digestion by
    Trypsin-ultra, Mass Spectrometry Grade for 16 hours, dried and subjected to MALDI-TOF MS.

    Product Source

    Isolated from bovine (Bos taurus) pancreas

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    Trypsin-ultra, Reaction Buffer-202X

    Advantages and Features

    Applications

    • Digestion of proteins for proteomic analysis by Mass Spectrometry
    • Protein and peptide identification

    Properties and Usage

    Reaction Conditions

    1X Trypsin-ultra, Reaction Buffer
    Incubate at 37°C

    1X Trypsin-ultra, Reaction Buffer:
    50 mM Tris-HCl
    20 mM CaCl2
    pH 8 @ 25°C

    Usage Concentration

    100 ng/μl

    Storage Temperature

    -20°C

    Molecular Weight

    Theoretical: 23675 daltons

    Specific Activity

    2.1 μmol/min/mg/

    Quality Control

    Quality Assurance Statement

    • Modified Trypsin 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 (Calcitonin Peptide Digestion):

      The endoproteinase is tested for its ability to digest a modified Human calcitonin peptide as determined by MALDI-TOF MS analysis.

    • 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. Substrate must be in phosphate-free buffer to prevent calcium precipitation with both reconstituted enzyme and enzyme buffer.
    2. Trypsin-ultra, Mass Spectrometry Grade is acetylated on multiple lysine residues. This protein appears as a single band on SDS-PAGE. This sequence is also available at www.neb.com.
    3. Reconstitution: Trypsin-ultra, Mass Spectrometry Grade should be reconstituted by the addition of 20–200 μl of high purity water. Rapid autolysis is a function of enzyme concentration.
    4. Storage: Supplied freeze-dried from a sodium acetate and calcium chloride buffer. Store at -20°C.
      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. Northrop, J.H. and Kunitz, M. (1931). Isolation of protein crystals possessing tryptic activity. Science. 73
    2. Perona, J.J. and Craik, C.S. (1995). Structural basis of substrate specificity in the serine proteases. Protein Sci.. 4, 337-360.

    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. I have a very low concentration of protein and would prefer not to denature as a separate step with buffer exchange before digestion. What denaturants can he use in the Trypsin reaction itself?
    3. I would like to use Trypsin to digest some proteins which are present in a cell culture supernatant. The supernatant is composed of DMEM supplemented with 0.2% BSA and antibiotics. Will the enzyme work in the cellular supernatant? The final reaction volume will be 100-300 ul.
    4. I am using Trypsin and am wondering about specificity. Does it cut at additional sites when in high concentration?
    5. I want to know if calcium can be left out of the buffer since it is causing my DNA-protein complex to precipitate.
    6. I want to use Trypsin on 20 ug of protein. How much enzyme do I need to use?
    7. What is your standard protocol for using this enzyme?
    8. The enzyme is supplied with 1X reaction buffer, but the reaction conditions call for 1X reaction buffer, so how should the reaction be set up?
    9. I diluted the Trypsin with 1X reaction buffer and found that it cannot be dissolved.
    10. What is the sequence for this Trypsin?
    11. Will Trypsin work in 50mM Ammonium Bicarbonate, pH 8.3 ?
    12. Regarding the specific activity reported for this enzyme, what substrate is being used in the assay?
    13. How does one do a Trypsin in-gel digest?
    14. Do any kind of specifications go along with this product?
    15. Is there a simple way to remove Trypsin after protein cleavage?
    16. Has there been any comparisons of our Trypsin against Roche's enzyme, Promega’s and products #25300054 & 25200056 from Invitrogen?
    1. Protocol using Trypsin-ultra™, Mass Spectrometry Grade (P8101)

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