PDF《P0703S》

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 add- ing

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 r Endo Hf P0703S 100,000 units 1,000,000 U/ml Lot:

0181407 RECOMBINANT Store at C20°C Exp: 7/16 Description: Endo Hf is a recombinant protein fusion of Endoglycosidase H and maltose binding protein. Endo Hf cleaves the chitobiose core of?high mannose and some hybrid oligosaccharides from N-linked glycoproteins (1) equally as well as Endo H. Source: Cloned from Streptomyces plicatus (2) and overexpressed in?E. coli?(3) Applications: ? Removal of high mannose N-glycans from glycoproteins 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. G5 Reaction Buffer, two-fold dilutions of Endo Hf are added and the reaction mix is incubated for

1 hour at 37°C. Separation of reaction products are visualized by SDS-PAGE. Specific Activity: ~232,000 units/mg. Molecular Weight: 70,000 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) (Man)n -Man xCMan y Man-GlcNAc-GlcNAcCAsnC C C C 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) Specificity: 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 add- ing

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 r Endo Hf P0703S 100,000 units 1,000,000 U/ml Lot: