324727 | Endoglycosidase F3, Elizabethkingia meningosepticum, Recombinant, E. coli

324727
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      Overview

      Replacement Information

      Key Specifications Table

      Pricing & Availability

      Catalog NumberAvailability Packaging Qty/Pack Price Quantity
      324727-200MIU
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          Plastic ampoule 200 miu
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          Description
          OverviewRecombinant, Elizabethkingia meningosepticum endoglycosidase F3 expressed in E. coli. Cleaves asparagine-linked or free biantennary and triantennary complex, and Man3GlcNAc oligosaccharides from glycoproteins. This enzyme cleaves between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine residue. Core fucosylation increases the activity of Endo F3 up to 40 fold. Exhibits no activity on high mannose and hybrid molecules. Less sensitive to protein conformation than N-Glycosidase F (Cat. No. 362185) and therefore is more suitable for deglycosylation of native proteins.
          Note: 1 mU = 1 milliunit.
          Catalogue Number324727
          Brand Family Calbiochem®
          SynonymsEndo-β-N-acetylglucosaminidase F3, Endo F3
          References
          ReferencesTarentino, A.L., et al. 1995. Glycobiology 5, 599.
          Tarentino, A.L., and Plummer, T.H. 1994. Methods Enzymol. 230, 44.
          Tarentino, A.L., et al. 1993. J. Biol. Chem. 268, 9702.
          Trimble, R.B., and Tarentino, A.L. 1991. J. Biol. Chem. 266, 1646.
          Product Information
          Activity≥5 units/ml
          Unit of DefinitionOne unit is defined as the amount of enzyme that will release N-linked oligosaccharides from 1.0 µmol porcine fibrinogen per min at 37°C, pH 5.5.
          EC number3.2.1.96
          FormLiquid
          FormulationIn 20 mM Tris-HCl, pH 7.5.
          Applications
          Biological Information
          Specific Activity≥30 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
          324727

          References

          Reference overview
          Tarentino, A.L., et al. 1995. Glycobiology 5, 599.
          Tarentino, A.L., and Plummer, T.H. 1994. Methods Enzymol. 230, 44.
          Tarentino, A.L., et al. 1993. J. Biol. Chem. 268, 9702.
          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.

          Revision19-June-2008 RFH
          SynonymsEndo-β-N-acetylglucosaminidase F3, Endo F3
          DescriptionRecombinant, Elizabethkingia meningosepticum endoglycosidase F3 expressed in E. coli. Cleaves asparagine-linked or free biantennary and triantennary complex, and Man3GlcNAc oligosaccharides from glycoproteins. This enzyme cleaves between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine residue. Core fucosylation increases the activity of Endo F3 up to 40 fold. Exhibits no activity on high mannose and hybrid molecules. Less sensitive to protein conformation than N-Glycosidase F (Cat. No. 362185) and therefore is more suitable for deglycosylation of native proteins.
          FormLiquid
          FormulationIn 20 mM Tris-HCl, pH 7.5.
          Recommended reaction conditions50 mM sodium acetate buffer, pH 4.5 Endoglycosidase F3, Chryseobacterium meningosepticum, Recombinant, E. coli 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. Endoglycosidase F3 cleaves asparagine-linked or free biantennary and triantennary complex oligosaccharides and Man3GlcNAc oligosaccharides from glycoproteins. It cleaves between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide, generating a truncated sugar molecule with one Nacetylglucosamine residue remaining on the asparagine residue. Core fucosylation increases the activity of the Endoglycosidase F3 up to 40-fold. Endoglycosidase F3 is less sensitive to protein conformation than N-Glycosidase F (Cat. No. 362185) and is, therefore, more suitable for deglycosylation of native proteins. Endo F3 is highly sensitive to the presence of detergents, so it is recommended that any residual detergents be removed prior to deglycosylation. Required Reagents Substrate: porcine fibrinogen (6.1 mg/ml) in 1X assay buffer Diluted enzyme preparation: dilute the endoglycosidase F3 1:400 in 1X assay buffer 5X assay buffer: 250 mM sodium acetate, pH 4.5 Triton® X-100 detergent (Cat. No. 648462): 15% solution SDS-PAGE system Protocol 1. Add 50 µl (304 µg) of substrate to each of 6 tubes. 2. Add 0, 1, 2, 3, 4, or 5 µl of diluted enzyme to appropriately labeled tubes and incubate 1 h at 37°C. 3. Stop the reaction by heating to 100°C for 5 min. 4. Run 10 µl of each sample on a 10% SDS-PAGE gel. Stain with Coomassie Blue. 5. Note the lowest tube number in which ~50% of the substrate is cleaved. 6. Calculate the activity: Units/ml = µmol/min/ml = (8 X 105) R/M T V R = 152 (µg of fibrinogen cleaved M = 185,000 (formula weight of fibrinogen) T = 60 (time in min) V = volume (µl) of enzyme dilution needed to cleave 50% For example: if 2 µl of diluted enzyme results in 50% cleavage, the activity would be 5.5 U/ml. Assay for Deglycosylation Required Reagents 5X assay buffer: 250 mM sodium acetate, pH 4.5 sterile dH2O glycoprotein sample Endoglycosidase F3 SDS-PAGE system Protocol 1. Add up to 200 µg of glycoprotein to an eppendorf tube and adjust the final volume to 37.5 µl with dH2O. 2. Add 10 µl of 5X assay buffer. 3. Add 2 µl of Endoglycosidase F3 to the reaction, mix well, and incubate for 1 h at 37°C. Note: When cleaving non-core fucosylated biantennary and triantennary N-linked oligosaccharides, increase the incubation time to 15 days. Many glycoproteins are contaminated with proteases. We advise adding suitable protease inhibitors if long duration incubations are anticipated. As an alternative to long duration incubations with Endoglycosidase F3 for removal of other classes of N-linked oligosaccharides, we suggest using our Glycoprotein Deglycosylation Kit (Cat. No. 362280). 4. Monitor cleavage by SDS-PAGE. Stain with Coomassie Blue.
          EC number3.2.1.96
          ContaminantsProteases: none detected
          Specific activity≥30 units/mg protein
          Activity≥5 units/ml
          Unit definitionOne unit is defined as the amount of enzyme that will release N-linked oligosaccharides from 1.0 µmol porcine fibrinogen per min at 37°C, pH 5.5.
          Storage +2°C to +8°C
          Do Not Freeze Yes
          Toxicity Standard Handling
          ReferencesTarentino, A.L., et al. 1995. Glycobiology 5, 599.
          Tarentino, A.L., and Plummer, T.H. 1994. Methods Enzymol. 230, 44.
          Tarentino, A.L., et al. 1993. J. Biol. Chem. 268, 9702.
          Trimble, R.B., and Tarentino, A.L. 1991. J. Biol. Chem. 266, 1646.