PDF《P0705S》

Applications: ? Removal of N-linked glycans from glycoproteins ? Preferred formulation for HPLC intensive methods 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 GlycoBuffer 2: [0.5 M Sodium Phosphate (pH 7.5 @ 25°C)] 10% NP-40 Optimal incubation times and enzyme concentrations must be determined empirically for a particular substrate. 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 10X GlycoBuffer 2,

2 ?l 10% NP-40, H2 O and 1C5 ?l PNGaseF. 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 (65?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 NP-40 and GlycoBuffer 2, two-fold dilutions of PNGase F are added and the reaction mix is incubated for 1?hour at 37°C. Separation of reaction products are visualized by SDS-PAGE. Quality Assurance: No contaminating exoglycosidase or Endoglycosidase F1, F2 or F3 activity could be detected. No contaminating proteolytic activity could be detected. Molecular Weight: 36,000 daltons. Quality Controls Glycosidase Assays: 5,000?units of PNGase F were incubated with 0.1 mM of fluorescently- 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. (See other side) PNGase F (Glycerol-free) P0705S 15,000 units Lot:

0421505 Exp: 5/17 500,000 U/ml Store at 4°C Do not freeze Description: Peptide: N-Glycosidase F, also known as PNGase F, is an amidase which supplied glycerol free for optimal performance in HPLC intensive methods. PNGase F cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccha- rides from N-linked glycoproteins (1). Source: PNGase F is purified from Flavobacterium meningosepticum (2). xCMan xCMan Man-GlcNAc-GlcNAc-AsnC C C PNGase F hydrolyzes nearly all types of N-glycan chains from glycopeptides/ proteins. [x = H or sugar(s)] Specificity: Applications: ? Removal of N-linked glycans from glycoproteins ? Preferred formulation for HPLC intensive methods 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 GlycoBuffer 2: [0.5 M Sodium Phosphate (pH 7.5 @ 25°C)] 10% NP-40 Optimal incubation times and enzyme concentrations must be determined empirically for a particular substrate. 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 10X GlycoBuffer 2,

2 ?l 10% NP-40, H2 O and 1C5 ?l PNGaseF. 4. Incubate reaction at 37°C for