362185 N-Glycosidase F, Elizabethkingia meningosepticum

362185
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      Overview

      Replacement Information

      Key Specifications Table

      Pricing & Availability

      Catalog NumberAvailability Packaging Qty/Pack Price Quantity
      362185-100U
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          Plastic ampoule 100 u
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          Description
          OverviewNative N-Glycosidase F from Elizabethkingia meningosepticum. Catalyzes the hydrolysis of asparagine-linked high mannose, as well as hybrid and complex oligosaccharides from glycoproteins. This enzyme will not cleave oligosaccharides containing α1,3-linked core fucose commonly found on plant glycoproteins. N-Glycosidase F will also fail to cleave if the asparagine to which the oligosaccharide is attached is not peptide-bonded to at least one amino acid residue at both the amino and carboxy termini.
          Catalogue Number362185
          Brand Family Calbiochem®
          SynonymsGlycopeptidase F, PNGase F
          References
          ReferencesMeans, R.E. and Desrosiers, R.C. 2000. J. Virol. 74, 11181.
          Fan, J.Q., and Lee, Y.C. 1997. J. Biol. Chem. 272, 27058.
          Tarentino, A.L., and Plummer, T.H. 1994. Methods Enzymol. 230, 44.
          Trimble, R.B., and Tarentino, A.L. 1991. J. Biol. Chem. 266, 1646.
          Product Information
          CAS number83534-39-8
          Activity≥4500 units/ml
          Unit of DefinitionOne unit is defined as the amount of enzyme that will catalyze the release of N-linked oligosaccharides from 1.0 nmol denatured ribonuclease B per min at 37°C, pH 7.5. Cleavage is monitored by SDS-PAGE. One unit of N-Glycosidase F is equal to 1 IUB milliunit.
          EC number3.5.1.52
          FormLiquid
          FormulationIn 50 mM NaCl, 20 mM Tris-HCl, 1 mM EDTA, pH 7.5.
          Applications
          Biological Information
          Specific Activity≥20,000 units/mg protein
          Physicochemical Information
          ContaminantsProteases: none detected.
          Dimensions
          Materials Information
          Toxicological Information
          Safety Information according to GHS
          Safety Information
          Product Usage Statements
          Storage and Shipping Information
          Ship Code Blue Ice Only
          Toxicity Standard Handling
          Storage +2°C to +8°C
          Do not freeze Yes
          Packaging Information
          Transport Information
          Supplemental Information
          Specifications

          Documentation

          SDS

          Title

          Safety Data Sheet (SDS) 

          Certificates of Analysis

          TitleLot Number
          362185

          References

          Reference overview
          Means, R.E. and Desrosiers, R.C. 2000. J. Virol. 74, 11181.
          Fan, J.Q., and Lee, Y.C. 1997. J. Biol. Chem. 272, 27058.
          Tarentino, A.L., and Plummer, T.H. 1994. Methods Enzymol. 230, 44.
          Trimble, R.B., and Tarentino, A.L. 1991. J. Biol. Chem. 266, 1646.
          Data Sheet

          Note that this data sheet is not lot-specific and is representative of the current specifications for this product. Please consult the vial label and the certificate of analysis for information on specific lots. Also note that shipping conditions may differ from storage conditions.

          Revision21-November-2008 RFH
          SynonymsGlycopeptidase F, PNGase F
          DescriptionNative N-Glycosidase F from Elizabethkingia meningosepticum. Catalyzes the hydrolysis of asparagine-linked high mannose, as well as hybrid and complex oligosaccharides from glycoproteins. This enzyme will not cleave oligosaccharides containing α1-3-linked core fucose commonly found on plant glycoproteins. N-Glycosidase F will also fail to cleave if the asparagine to which the oligosaccharide is attached is not peptide-bonded to at least one amino acid residue at both the amino and carboxy termini.
          FormLiquid
          FormulationIn 50 mM NaCl, 20 mM Tris-HCl, 1 mM EDTA, pH 7.5.
          Recommended reaction conditions
          Note: This protocol is provided only as a general guideline. Researchers should standardize this assay for their own specific needs and should consult published literature. N-glycosidase F cleaves asparagine-linked high mannose, as well as hybrid and complex oligosaccharides, from glycoproteins. It deaminates the asparagine to aspartic acid, but leaves the oligosaccharide intact. N-glycosidase F will not remove oligosaccharides containing α1,3-linked core fucose commonly found on plant glycoproteins. In addition, N-glycosidase F will not cleave if the asparagine to which the oligosaccharide is linked is not peptide bonded to at least one amino acid residue at both the N- and C-termini. Detergent and heat denaturation increase the rate of cleavage up to 100X. Most native proteins can still be completely N-deglycosylated but the incubation time must be increased. N-glycosidase F will remain active under the recommended incubation conditions for at least 72 h. Assay for Unit Definition One unit of N-glycosidase F is defined as the amount of enzyme that will catalyze the release of N-linked oligosaccharides from 1 nmol of denatured Ribonuclease B per min at 37°C, pH 7.5. Cleavage is monitored by SDS-PAGE. One unit of Nglycosidase F is equal to 1 IUB milliunit. Required Reagents Substrate: Ribonuclease B at a concentration of 1.1 mg/ml in 1X assay buffer Enzyme: Dilute the preparation of N-glycosidase F 1:400 in 1X assay buffer 5X Assay Buffer: 250 mM NaHPO4, pH 7.5 Denaturation buffer: 2% SDS, 1 M β-mercaptoethanol (β-ME) dH2O SDS-PAGE system Triton® X-100 detergent (Cat. No. 648463) Protocol 1. Add 25 µl of denaturation buffer to 0.45 ml of substrate solution and heat to 100°C for 10 min. Cool and add 25 µl of Triton® X-100 detergent. 2. Add 50 µl (50 µg) of denatured substrate to each of 6 tubes. 3. Add 0, 1, 2, 3, 4, or 5 µl of diluted enzyme to appropriately labeled tubes and incubate 1 h at 37°C. 4. Stop the reaction by heating to 100°C for 5 min. 5. Run 10 µl of each sample on a 12% SDS-PAGE gel. Stain with Coomassie Blue. Ribonuclease B is ~15 kD. Note the lowest tube number in which ~50% of the substrate is cleaved. 6. Calculate the activity: Units/ml = (4 X 108) R\M T V R = 25 (µg of Ribonuclease B cleaved) M = 15,000 (formula weight of Ribonuclease B) T = 60 (time in min) V = volume (µl) of enzyme dilution needed to cleave 50% For example, if 2 µl of enzyme resulted in 50% cleavage, the activity would be 5,540 units/ml. Assay for Deglycosylation Required Reagents Glycoprotein substrate N-glycosidase F 5X Assay Buffer: 250 mM NaHPO4, pH 7.5 Denaturation buffer: 2% SDS, 1 M β-mercaptoethanol (β-ME) dH2O SDS-PAGE system Triton® X-100 detergent (Cat. No. 648463) Protocol 1. Add up to 200 µg of glycoprotein to an eppendorf tube. Adjust the final volume to 35 µl with dH2O. 2. Add 10 µl of 5X assay buffer and 2.5 µl of denaturation buffer. Heat to 100°C for 5 min. 3. Cool the tube and add 2.5 µl of Triton® X-100 detergent and mix. Note: Failure to add Triton® X-100 detergent will result in a 3-fold reduction of N-glycosidase F activity. 4. Add 2 µl of N-glycosidase F to the reaction. Incubate for 3 h at 37°C. Note: If SDS or heat denaturation is omitted, it may be necessary to increase the incubation time to at least 24 h 5. Monitor the cleavage by SDS-PAGE.
          CAS number83534-39-8
          EC number3.5.1.52
          ContaminantsProteases: none detected.
          Specific activity≥20,000 units/mg protein
          Activity≥4500 units/ml
          Unit definitionOne unit is defined as the amount of enzyme that will catalyze the release of N-linked oligosaccharides from 1.0 nmol denatured ribonuclease B per min at 37°C, pH 7.5. Cleavage is monitored by SDS-PAGE. One unit of N-Glycosidase F is equal to 1 IUB milliunit.
          Storage +2°C to +8°C
          Do Not Freeze Yes
          Toxicity Standard Handling
          ReferencesMeans, R.E. and Desrosiers, R.C. 2000. J. Virol. 74, 11181.
          Fan, J.Q., and Lee, Y.C. 1997. J. Biol. Chem. 272, 27058.
          Tarentino, A.L., and Plummer, T.H. 1994. Methods Enzymol. 230, 44.
          Trimble, R.B., and Tarentino, A.L. 1991. J. Biol. Chem. 266, 1646.