PDF《P0705S》

2 ?l 10% NP40, 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 G7?Reaction Buffer, 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:

0391302 Exp: 2/15 500,000 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: y New Quality Controls P0705S

039130215021 P0705S

039130215021 Page

2 (P0705) 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: Neu5Acα2-3Galβ1-3GlcNAcβ1-3 Galβ1-4Glc-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 F1 , F2 , H: Dansylated invertase high mannose. ND Endo F2 , F3 : Dansylated fibrinogen biantennary. ND Endoglycosidase F1 Assay: After incubation of 5,000 units of PNGase F with

20 pmol of 2-AA Man-5 fluorescent standard, for

20 hours at 37°C, no endoglycosidase F1 activity could be detected by LC/MS analysis with fluorescence detection. Protease Assay: After incubation of 10,000 units of PNGase F 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. Physical Purity: Purified to > 95% homogene- ity as determined by SDS-PAGE analysis using Coomassie Blue detection. Heat Inactivation:

500 units of enzyme were inactivated by incubation at 75°C for

10 minutes. Notes: Since PNGase F activity is inhibited by SDS, it is essential to have NP-40 present in the reaction mixture. Why this non-ionic detergent counteracts the SDS inhibition is unknown at present. To deglycosylate a native glycoprotein, longer incubation time as well as more enzyme may be required. PNGase F will not cleave N-linked glycans con- taining core α1-3 Fucose. Previously supplied as a recombinant. Repeated freeze thaw cycles degrade enzyme activity over time. References: 1. Maley, F. et al. (1989) Anal. Biochem. 180, 195C204. 2. Plummer, T.H., Jr. and Tarentino, A.L. (1991) Glycobiology 1, 257C263. Companion Product: RNase B (NEB #P7817S) Page