PDF《Endo H》

1-800-632-7799 i n f o @ n e b.

c o m w w w. n e b. c o m Endo H P0702S 10,000 units 500,000 U/ml Lot:

0161210 RECOMBINANT Store at C20°C Exp: 10/14 Description: Endoglycosidase H is a recombinant glycosidase which cleaves the chitobiose core of high mannose and some hybrid oligosaccharides from N-linked glycoproteins (1) Reaction Conditions: Typical reaction conditions are as follows: 1. Combine 1C20 ?g of glycoprotein,

1 ?l of 10X Glycoprotein Denaturing Buffer and H2 O (if necessary) to make a

10 ?l total reaction volume. 2. Denature glycoprotein by heating reaction at 100°C for

10 minutes. 3. Make a total reaction volume of

20 ?l by adding

2 ?l of 10X G5 Reaction Buffer, H2 O and 1C5 ?l Endo H. 4. Incubate reaction at 37°C for

1 hour. Note: Reactions may be scaled-up linearly to accommodate larger reaction volumes. Unit Definition: One unit is defined as the amount of enzyme required to remove >

95% of?the carbohydrate from

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: Endo H P0702S 10,000 units 500,000 U/ml Lot:

0161210 RECOMBINANT Store at C20°C Exp: 10/14 Description: Endoglycosidase H is a recombinant glycosidase which cleaves the chitobiose core of high mannose and some hybrid oligosaccharides from N-linked glycoproteins (1) Reaction Conditions: Typical reaction conditions are as follows: 1. Combine 1C20 ?g of glycoprotein,

1 ?l of 10X Glycoprotein Denaturing Buffer and H2 O (if necessary) to make a

10 ?l total reaction volume. 2. Denature glycoprotein by heating reaction at 100°C for

10 minutes. 3. Make a total reaction volume of

20 ?l by adding

2 ?l of 10X G5 Reaction Buffer, H2 O and 1C5 ?l Endo H. 4. Incubate reaction at 37°C for

1 hour. Note: Reactions may be scaled-up linearly to accommodate larger reaction volumes. Unit Definition: One unit is defined as the amount of enzyme required to remove >

95% of?the carbohydrate from