Endoglycosidase H -ME). Heat β sodium mM -mercaptoethanol β M of the Streptomyces endoglycosidase H in of the Streptomyces Escherichia coli. Biol Chem 256:10640-10644 (1981). Gibson, N. Royal and R. Das, B. W. Maley, Maley , G. F. of the Streptomyces K. Biemann. Primary structure enzyme endo-beta-N-acetylglucosaminidase H. J Biol Chem 259:7577-7583 (1984). 5X Reaction buffer 5.5- 250 5X Reaction buffer phosphate pH 5.5 Denaturation solution- 2% w/v sodium lauryl sulfate, 1 tube. Adjust to 37.5 µL final volume with deionized water. 2.5 µl of Denaturation Solution (SDS/ at 100°C for 5 minutes. X-100. SDS NOTE: It is not necessary to add Triton will not inactivate Endoglycosidase H. Incubate 3 hours at 37°C. reaction. 1. Wirth and C. J. Hering. Expression D. F. W., Robbins P. 2. Wirth, C. Hering, F. D. F. R. B. Trimble, W., Robbins P. ¾ ¾ 1. to Eppendorf 200 µg of Add up to 2. and 5X Endoglycosidase H Buffer Add 10 µL 3. Cool. Add 2.0 µL of Endoglycosidase H to the Procedure for Deglycosylation Procedure Specificity or high oligo- -linked hybrid saccharides. as One unit of Endoglycosidase H activity is defined to catalyze the the amount of enzyme required 1 µmole of of N-linked from release Ribonuclease B in 1 minute at 37°C, pH denatured (cleaved by SDS-PAGE 7.5. Cleavage is monitored Ribonuclease B migrates faster). If SDS or heat denaturation is omitted, it may be incubation time. necessary to increase References Reagents Suggestions for Use Assay mM Streptomyces EDTA. EDTA. mM -N-acetylglucosaminidase β NaCI, 1 mM 5 U/mL ∼ clone. Purity Stability properly. Stable at least 12 months when stored will to ambient temperature Several days exposure activity. not reduce Product Description Product Specifications Molecular Weight 29,000 Daltons Endoglycosidase H is tested for contaminating BSA is as follows; 10 µg of denatured protease incubated for 24 hours at 37°C with 2 µL of enzyme. BSA shows no analysis of the treated SDS-PAGE evidence of degradation. host strain has been extensively The production any detectable tested and does not produce glycosidases. Activity 40 U/mg, Endoglycosidase H [Endo- Storage at 4°C. Do Not Freeze Store Formulation as a sterile solution in 20 The enzyme is provided Tris HCI pH 7.5, 50 Tris H, EC 3.2.1.96] cleaves asparagine-linked oligo- H, EC 3.2.1.96] cleaves not complex, oligo- mannose and hybrid, but 1). It (see Figure saccharides from N-acetylglucosamine cleaves between the two of the in the diacetylchitobiose core residues , generating a truncated sugar molecule with one N-acetylglucosamine residue on the asparagine. In contrast, PNGase F remaining the oligosaccharide intact. Detergent and removes the rate of cleavage heat denaturation may increase for some glycoproteins. a from Endoglycosidase H is produced plicatus Endoglycosidase H Endoglycosidase Endoglycosidase H 60 µL +4 Package Size Temp.°C Nacetylglucosaminidase H from Streptomyces plicatus. Streptomyces from Nacetylglucosaminidase H (1987). Methods Enzymol 138:763-770 of Amplified expression Maley and R. B. Trimble. endo-beta-N-acetylglucosaminidase H in Streptomyces of the enzyme Escherichia coli and characterization J Biol Chem 260:5683-5690 (1985). product. 5. Endo-beta- Maley. and F. R. B., R. J. Trumbly Trimble 6. F. D. Ziegler, M. Belfort, F. Robbins, W. J., P. R. Trumbly ) r r PNGase F PNGase F recombinant r (1-4) GlcNAc-N-Asn β r Endo H (1-4) GlcNAc-N-Asn β Endo H (1-4) GlcNAc β (1-4) GlcNAc β

Endoglycosidase H (Streptomyces plicatus r (1-3) (1-6) α α (1-3) (1-6) α α (1-4) Man Man β Cleavage of oligosaccharides by Endoglycosidase H and PNGase F Cleavage of oligosaccharides by Endoglycosidase (1-6) (1-3) Man Man - α α (1-2) Man β (1-6) (1-3) Man α α GlcNAc Maley. Endo-beta-N-acetylglucosaminidase L from Endo-beta-N-acetylglucosaminidase Maley. plicatus. Methods Enzymol 83:603-610 Streptomyces (1982). by endo-beta- linked high-mannose oligosaccharides Anal Biochem 141:515-522 Nacetylglucosaminidase H. (1984). Man Man Hybrid type Oligomannose type Man Man GlcNAc Order Information Order 100467-1 Catalog No. Description Product 3. and F. , G. E Aumick R. B., A. L. Tarentino Trimble 4. of N- Optimizing hydrolysis Maley. R. B. and F. Trimble Man = Mannose; GlcNAc = N-acetylglucosamine Figure 1 Figure PN001548 Rev002