PDF《Endo H》

10 ?g of denatured RNase?B in

1 hour at 37°C in a total reaction volume of

10 ?l (10?NEB units =

1 IUB milliunit). Unit Definition Assay:

10 ?g of RNase B are denatured with 1X Glycoprotein Denaturing Buffer at 100°C for 10?minutes. After the addition of 1X G5 Reaction Buffer, two-fold dilutions of Endo H r are added and the reaction mix is incubated for

1 hour at 37°C. Separation of reaction products is visualized by SDS-PAGE. Specific Activity: ~ 915,000 units/mg Molecular Weight: 29,000 daltons Quality Assurance: No contaminating exoglycosidase or proteolytic activity could be detected. Quality Controls Glycosidase Assays: 5,000 units of Endo Hwere incubated with 0.1 mM of flourescently-labeled oligosaccharides and glycopeptides, in a 10??l reaction for

20 hours at 37°C. The reaction products were analyzed by TLC for digestion of substrate. Physical Purity: Purified to >

95% homogeneity as determined by SDS-PAGE analysis using Coomassie Blue detection. (See other side) Source: Cloned from Streptomyces plicatus (2) and overexpressed in?E.?coli?(3). Applications: ? Removal of carbohydrate residues from proteins Supplied in:

50 mM NaCl,

20 mM Tris-HCl (pH?7.5 @ 25°C) and

5 mM Na2 EDTA. Reagents Supplied with Enzyme: 10X Glycoprotein Denaturing Buffer: 5% SDS, 0.4 M DTT 10X G5 Reaction Buffer: 0.5 M Sodium Citrate (pH 5.5 @ 25°C) Optimal incubation times and enzyme concentrations must be determined empirically for a particular substrate. (Man)n -Man Man-GlcNAc-GlcNAcCAsnC Endo H and Endo Hf cleave only high mannose structures (n = 2C150, x = (Man)1C2 , y = H) and hybrid structures (n = 2, x and/or y = AcNeu-Gal-GlcNAc) xCMan y C C C Specificity: P0702S

016121014101 P0702S

016121014101 Page

2 (P0702) No other glycosidase activities were detected (ND) with the following substrates: β-N-Acetyl-glucosaminidase: GlcNAcβ1-4GlcNAcβ1-4GlcNAc-AMC ND α-Fucosidase: Fucα1-2Galβ1-4Glc-AMC Galβ1-4 (Fucα1-3)GlcNAcβ1-3Galβ1-4Glc-AMC ND β-Galactosidase: Galβ1-3GlcNAcβ1-4Galβ1-4Glc-AMC ND α-Galactosidase: Galα1-3Galβ1-4Galα1-3Gal-AMC ND α-Neuraminidase: Neu5Acα2-3Galβ1-3GlcNAcβ1-3Galβ 1-4Glc-AMC ND α-Mannosidase: Manα1-3Manβ1-4GlcNAc-AMC Manα1-6Manα1-6(Manα1-3)Man-AMC ND β-Glucosidase: Glcβ1-4Glcβ1-4Glc-AMC ND β-Xylosidase: Xylβ1-4Xylβ1-4Xylβ1-4Xyl-AMC ND β-Mannosidase: Manβ1-4Manβ1-4Man-AMC ND Endo F2 , F3 : Dansylated fibrinogen biantennary. ND PNGase F: Fluoresceinated fetuin triantennary. ND Protease Assay: After incubation of 5,000 units of Endo H with 0.2 nmol of a standardized mixture of proteins, for 20?hours at 37°C, no proteolytic activity could be detected by SDS-PAGE. Notes On Use: Enzymatic activity is not affected by SDS. To deglycosylate a native glycoprotein, longer incubation time as well as more enzyme may be required. References: 1. Maley, F. et al. (1989) Anal. Biochem. 180, 195C204. 2. Robbins, P. et al. (1984) J. Biol. Chem. 259, 7577C7583. 3. Guan, C and Wong, S., New England Biolabs Inc., unpublished results. Companion Product: RNase B (NEB #P7817S) Page

2 (P0702) β-N-Acetyl-glucosaminidase: GlcNAcβ1-4GlcNAcβ1-4GlcNAc-AMC ND α-Fucosidase: Fucα1-2Galβ1-4Glc-AMC Galβ1-4 (Fucα1-3)GlcNAcβ1-3Galβ1-4Glc-AMC ND β-Galactosidase: Galβ1-3GlcNAcβ1-4Galβ1-4Glc-AMC ND α-Galactosidase: Galα1-3Galβ1-4Galα1-3Gal-AMC ND α-Neuraminidase: Neu5Acα2-3Galβ1-3GlcNAcβ1-3Galβ 1-4Glc-AMC ND α-Mannosidase: Manα1-3Manβ1-4GlcNAc-AMC Manα1-6Manα1-6(Manα1-3)Man-AMC ND β-Glucosidase: Glcβ1-4Glcβ1-4Glc-AMC ND β-Xylosidase: Xylβ1-4Xylβ1-4Xylβ1-4Xyl-AMC ND β-Mannosidase: Manβ1-4Manβ1-4Man-AMC ND Endo F2 , F3 : Dansylated fibrinogen biantennary. ND PNGase F: Fluoresceinated fetuin tri........