Endo-H (C09-0882-162)

Product DescriptionEndo H, endo-beta-N-acetylglucosaminidase H, Endoglycosidase HEndo H cleaves Asparagine-linked hybrid or high mannose oligosaccharides, but not complex oligosaccharides. It 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. In contrast, PNGase F removes the oligosaccharide intact. Detergent and heat denaturation may increase the rate of cleavage for some glycoproteins.Recommended Reagentsincluded with:1 vial: 5x Reaction Buffer - 400 ml250 mM sodium phosphate, pH5.51 vial: Denaturation Solution - 200 ml 2%SDS, 1 M Beta-mercaptoethanol Molecular weightapproximately 29 kDpH optimum:5.5, active over the range 5-6.ApplicationsReleases asparagine-linked hybrid or high mannose oligosaccharides, but not complex oligosaccharides. Endo H cleaves between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosac-charide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. In contrast, PNGase F removes the oligosaccharide intact. Detergent and heat denaturation may increase the rate of cleavage for some glycoproteins.Specific ActivityOne unit of Endo H activity is defined as the amount of enzyme required to catalyze the release of N-linked oligosaccharides from 1 µmole of denatured Ribonuclease B in one minute at 37˚C, pH 5.5. Cleavage is monitored by SDS-PAGE (cleaved Ribonuclease B migrates faster). SpecificityEndo H cleaves Asparagine-linked hybrid or high mannose oligosaccharides, but not complex oligosaccharides. It 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. In contrast, PNGase F removes the oligosaccharide intact. Detergent and heat denaturation may increase the rate of cleavage for some glycoproteins.FormulationThe enzyme is provided as a sterile-filtered solution in 20 mM Tris-HCl, 25mM NaCl, 1 mM EDTA (pH 7.5).StabilitySeveral days exposure to ambient temperatures will not reduce activity. Stable at least 12 months when stored properly. Quality & PurityEndo H is tested for contaminating protease as follows: 10 µg of denatured BSA is incubated at 37°C for 24 hours with 2 µl of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation. The production host strain has been extensively tested and does not produce any detectable glycosidases.Directions for use1. Add up to 200 µg of glycoprotein to an Eppendorf tube. Adjust to 37.5 µl final volume with de-ionized water.2. Add 10 µl 5x Reaction Buffer 5.5 and 2.5 µl of Denaturation Solution. Heat at 100°C for 5 minutes.NOTE: It is not necessary to add Triton X-100. SDS will not inactivate Endo H.3. Add 2.0 µl of Endo H to the reaction. Incubate 3 hours at 37°C.If SDS or heat denaturation is omitted, increase incubation time to at least 24 hours.Monitor cleavage by SDS-PAGE.The production host strain has been extensively tested and does not produce any detectable glycosidases. Specifications and Purity: Specific Activity >40 U/mg;Activity >5 U/ml. Related Document: https://ald-pub-files.oss-cn-shanghai.aliyuncs.com/aladdinsci/pdp/sds/1/E489833-SCI_3100ed06d19a94b3ff9746ae4406ddcc.pdf.