Alpha-(1-3,6)-Galactosidase. Specifications - Protocol. Recommended Reagents included with E-AG07: Reaction buffer - 250mM Sodium phosphate, ph 6.

Transcription

1 Alpha-(1-3,6)-Galactosidase Source Recombinant from E. Coli in E.Coli Catalog Number E-AG02 60 µl E-AG µl E-AG µl Recommended Reagents included with E-AG07: Reaction buffer - 250mM Sodium phosphate, ph 6.5 Specific Activity 30 U/mg Activity 400 U/ml Formulation The enzyme is provided as a sterile-filtered solution in 50 mm sodium phosphate, ph 7.5 Specificity Non-reducing terminal alpha-(1-3)- and alpha-(1-6)-galactose. There is no activity on alpha-(1-4)-galactose. Properties molecular ~80,000 daltons Applications Structural analysis of oligosaccharides Xenograft transplantation studies Removing heterogeneity from glycoproteins Specific Activity One unit of alpha-(1-3,6) Galactosidase is defined as the amount of enzyme required to produce 1 µmole of p-nitrophenol (pnp) in 1 minute at 25 C ph 6.5 from p-nitrophenyl-alpha-dgalactopyranoside. Purity Each lot of alpha-(1-3,6)-galactosidase is tested for contaminating activities by incubating the enzyme for 24 hours with the appropriate substrates; the detection limit of these assays is 5 µu/ml (IUB). A passing lot will have no detectable activity. For the protease assay, 10 µg of denatured BSA is incubated for 24 hours with 2 µl of enzyme. Analysis of the BSA band after SDS-PAGE should show no evidence of degradation. Stability Stable at least 12 months when stored properly. Several days exposure to ambient temperatures will not reduce activity. Storage Store enzyme at 4 C. Do not freeze. Alpha-(1-3,6)-Galactosidase Specifications - Protocol

2 Directions for use 1. Add up to 100 µg of asialoglycoprotein or 1 nmol of oligosaccharide to tube. 2. Add water to 14 µl and 4 µl 5X Reaction Buffer. 3. Add 2 µl alpha-(1-3,6)-galactosidase. 4. Incubate at 37 C for 1 hour. Longer incubations are necessary if fucose is present on the penultimate sugar. References Kobata, A. Use of endo- and exoglycosidases for structural studies of glycoconjugates. Anal Biochem 100: 1-14 (1979). Prime, S. J. Dearnley, A.M. Venton, R.B. Parekh and C.J. Edge. Oligosaccharide sequencing based on exo- and endoglycosidase digestion and liquid chromatographic analysis of the products. J Chromatogr A 720: (1996) Dwek, R.A., C.J. Edge, D.J. Harvey, M.R. Wormald and R.B. Parekh. Analysis of glycoprotein-associated oligosaccharides. Ann Rev Biochem 62: Schmid K, Schmitt R. Raffinose metabolism in Escherichia coli K12. Purification and properties of a new alphagalactosidase specified by a transmissible plasmid. Eur J Biochem: 67(1): (1976) E-AG02 Product Specifications - Protocol Warranties and liabilities warrants that the above product conforms to the specifications described herein. Should the product fail for reasons other than through misuse will, at its option, replace free of charge or refund the purchase price. This warranty is exclusive and makes no other warrants, expressed or implied, including any implied conditions or warranties of merchantability or fitness for any particular purpose. For research use only Updated October 3, 2012

3 Sialic Acid Aldolase Source produced from a Escherichia coli strain K1 clone. Catalog Number E-ALD01 Certification of Analysis Lot Number 912.1B Contents 1 vial: Sialic Acid Aldolase- 60 µl (6 U) in 20 mm tris-hcl, ph 7.5 Specific Activity ~15 U/mg Activity ~100 U/ml Formulation The enzyme is provided as a sterile-filtered solution in 20 mm Tris-HCl, ph 7.5 Specificity Sialic Acid Aldolase (N-Acetylneuraminate pyruvate lyase, EC ) catalyzes the reversible reaction of sialic acid (N-acetylneuraminic acid) to N-acetylmannosamine and pyruvic acid. Molecular weight ~32,000 daltons Stability Stable at least 12 months when stored properly. Several days exposure to ambient temperatures will not reduce activity. Storage Store enzyme at 4 C. Do not freeze. Applications The enzyme is found in several bacterial strains which use the reverse reaction to degrade N-acetylneuraminic acid (sialic acid).the forward reaction is particularly useful for the determination of sialic acid concentrations by quantitatively converting it to N-acetylmannosamine and pyruvate. Since sialic acid is both negatively charged and a nonreducing sugar, its direct analysis is more difficult than conventional sugars. N-acetylmannosamine, however, can be assayed as a conventional reducing sugar by various techniques such as flourescent dye or radioactive labeling. Alternatively, the pyruvic acid generated in the reaction can be assayed using enzymes such as Lactic Dehydrogenase, coupled to NADH oxidation, to reduce pyruvate. NADH oxidation can be spectrophotometrically quantitated. Another method uses pyruvate oxidase to generate hydrogen peroxide which is measured colorimetrically. In addition to free neuraminic acid, N-acetylneuraminic Acid Aldolase can be used to determine the total amount of neuraminic acid in: Glycoproteins Cell surfaces Polysialic acids Capsular Polysaccharides by first digesting the whole cells, glycoprotein or polysaccharide with Sialidase Au (E-S001), and then determining total N-acetylneuraminic acid E-ALD01 Sialic Acid Aldolase Specifications - Protocol info@.com fax

4 E-ALD01 Product Specifications - Protocol Activity One unit of N-acetylneuraminic Acid Aldolase will release one µmole of pyruvate from N-acetylneuraminic acid in one minute at 37 C, ph 7.5 at a substrate concentration of 20 mm. Pyruvate production is monitored by the oxidation of NADH in the presence of Lactic Dehydrogenase. Purity Each lot of N-acetylneuraminic Acid Aldolase is tested for contaminating NADH Oxidase by incubating the enzyme for 24 hours at 37C with the appropriate substrate; the detection limit of this assay is 5 µu/ml (IUB). A passing lot will have no detectable activity. For the protease assay, 10 µg of denatured BSA is incubated for 24 hours with 2 µl of enzyme. Analysis of the BSA band after SDS-PAGE should show no evidence of degradation. Directions for use Assay Setup First, obtain an approximation of the amount of sialic acid (NANA) in the sample to be quantitated (from the literature or gel analysis after neuraminidase treatment). Otherwise run a series of dilutions to determine the quantity of sample to be assayed. In order to meet the volumetric requirements of the assay, diluted samples may be concentrated Assay of Free Sialic Acid Overview NANA is converted to pyruvic acid by N-acetylneuramanic acid Aldolase, and then subsequently treated with Lactic Dehydrogenase to form lactic acid with the oxidation of β NADH to β-nad (monitored spectrophotometrically). Additonal Reagents NANA Sample - The sample may be in solution or a lyophilizate that when added to the Reaction Buffer should contain between 1 and 200 nmoles of NANA. 25 mm Tris Reaction Buffer Lactic Dehydrogenase β-nadh Solution - Just prior to use, add 256 µl Tris Reaction Buffer to one of the vials of β-nadh (supplied with the kit) to make a 0.01 M solution (A340 = 62.2). Store at 40C in the dark for not more than 1 month. Discard if A340 drops 20%, or solution turns yellow. Procedure 1. Add sample to Tris Reaction Buffer so that the final volume is 980 µl and equilibrate to 37 C. 2. Add 1 µl Aldolase. Incubate in a 37 C water bath for a minimum of 10 minutes. 3. Pipette entire contents into cuvette and blank the spectrophotometer. Add 20 µl b-nadh Solution and mix by inverting several times. 4.Incubate in a 37C water bath for a minimum of 10 minutes. 5.Read and record the A initial. The A340 should read ~ Incubate in a 37EC water bath for a minimum of 10 minutes. 7.Read and record the A final. Calculate the nmoles of NANA: nmoles NANA= ((A initial -A final )x1000)/6.22 References Kolisis, F.N. An immobilized bienzyme system for assay of sialic acid. Biotechnol Appl Biochem 8:). Simpson, H., G. D. Ghusney, M. A. Crook and J. C. Pickup. Serum sialic acid enzymatic assay based on microtitre plates: application for measuring capillary serum sialic acid concentrations. Br J Biomed Sci 50: (1993). Sugahara K, K. Sugimoto, O. Nomura and T. Usui. Enzymatic assay of serum sialic acid. Clin Chim Acta 108:). Lilley, G.G., M. von Itzstein and N. Ivancic. High- Level Production and Purification of Escherichia coli N-Acetylneuraminic Acid Aldolase (EC ) Protein Expression and Purification 3: (1992). Ohta,Y., K. Watanabe and A. Kimura. Complete Sequence of E. coli N-acetylneuraminate lyase. Nucleic Acids Res.13: (1985). Warranties and liabilities warrants that the above product conforms to the specifications described herein. Should the product fail for reasons other than through misuse will, at its option, replace free of charge or refund the purchase price. This warranty is exclusive and makes no other warrants, expressed or implied, including any implied conditions or warranties of merchantability or fitness for any particular purpose. For research use only Updated October 4, info@.com fax

5 Alpha-(1-2,3,6)-Mannosidase Alpha-D-Mannoside Mannohydrolase Source Jack Bean Catalog Number E-AM01 60 µl E-AM µl E-AM µl EC Recommended Reagents included with E-AM01: 1 vial: 5x Reaction buffer mm sodium phosphate ph µl Activity 9 U/ml Specific activity 4 U/mg Specific Activity One unit of Alpha-(1-2,3,6)-Mannosidase is defined as the amount of enzyme required to hydrolyze 1 µmole of p-nitrophenyl-alpha-p-mannoside to p-nitrophenol in 1 minute at ph 5.0 and 37 C. Specificity Cleaves all Alpha-(1-2,3,6)-linked mannose. Formulation The enzyme is provided as a sterile-filtered solution in 20 mm Tris ph 7.5, 50 mm NaCl, 0.1 mm zinc chloride. Stability Several days exposure to ambient temperatures will not reduce activity. Stable at least 12 months when stored properly. Quality & Purity α-mannosidase is tested for contaminating protease as follows: 10 µg of denatured BSA is incubated at 37 C for 24 hours with 2 µl of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation. Enzymes purified from native sources are tested for contaminating exoglycosidases. The absence of exoglycosidase contaminants is confirmed by extended incubations with the corresponding pnp-glycosides. Molecular Weight two polypeptides of 44 and 64 kd ph optimum: 5 Storage Store enzyme at 4 C. Do not freeze. Alpha(1-2,3,6) Mannosidase Specifications - Protocol

6 Directions for use E-AM01 Product Specifications - Protocol 1. Add up to 1 nm of oligosaccharide to tube. 2. Add water to 15 µl 3. Add 4 µl 5x Reaction Buffer. 4. Add 1.0 µl of Mannosidase to the reaction. Incubate 10 minutes at 37 C. Warranties and liabilities warrants that the above product conforms to the specifications described herein. Should the product fail for reasons other than through misuse will, at its option, replace free of charge or refund the purchase price. This warranty is exclusive and makes no other warrants, expressed or implied, including any implied conditions or warranties of merchantability or fitness for any particular purpose. shall not be liable for any incidental, consequential or contingent damages. This product is intended for in vitro research only. revised on May 28, 2018

7 α-(1-6) Core Mannosidase α-d-mannosidase Mannohydrolase Source recombinant from Streptococcus pneumoniae in E. Coli Catalog Number E-AM02 60 µl E-AM µl E-AM µl EC Recommended Reagents included with E-AM02: 1 vial: 5x Reaction buffer 250 mm NaHPO 4, ph 5 Activity 1 U/ml Specific Activity 0.75 U/mg Application Analysis of mannose linkages Removal of α-(1-6) mannose resistant to other mannosidase enzymes Molecular Weight ~52,000 daltons Storage Store enzyme at 4 C. Do not freeze. Specific Activity One unit of α-(1-6) Core Mannosidase is defined as the amount of enzyme required to produce 2 µmoles of p-nitrophenol (pnp) in 1 minute at 37 C, ph 5.0 from α-(1-6) mannobiose. Specificity Cleaves unbranched non-reducing terminal mannose, α(1-6) linked to the beta-linked core mannose of the conserved mannosylchitobiose core of N-linked oligosaccharides. The presence of fucose linked to the core N-acetylglucosamine has no effect on cleavage. The enzyme may inhibit other mannosidases if a noncleavable α(1-6) mannose is present on the substrate. It should therefore always be added subsequent to digestion by other mannosidases. Formulation The enzyme is provided as a sterile-filtered solution in 50 mm Sodium phosphate 0.1 mm ZnCl 2 ph 7.5. Stability Stable at least 12 months when stored properly. Several days exposure to ambient temperatures will not reduce activity. Purity α-(1-6)-mannosidase is tested for contaminating protease as follows: 10 µg of denatured BSA is incubated at 37 C for 24 hours with 2 µl of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation. The production host strain has been extensively tested and does not produce any detectable glycosidases. α-(1-6)-mannosidase Specifications - Protocol

8 E-AM02 Product Specifications - Protocol Directions for use 1. Add up to 1 nmol of oligosaccharide to tube. 2. Add de-ionized water to a total of 15 µl. 3. Add 4 µl 5x Reaction Buffer Add 1 µl α-(1-6) Core Mannosidase. 5. Incubate at 37 C for 10 minutes. Progress may be monitored by SDS-PAGE if the size differential between native and de-glycosylated protein is sufficient for detection. Warranties and liabilities, inc warrants that the above product conforms to the specifications described herein. Should the product fail for reasons other than through misuse, LLC will, at its option, replace free of charge or refund the purchase price. This warranty is exclusive and, LLC makes no other warrants, expressed or implied, including any implied conditions or warranties of merchantability or fitness for any particular purpose., LLC shall not be liable for any incidental, consequential or contingent damages. This product is intended for in vitro research only. revised on May 28, 2018

9 ß-(1-3,4,6)-Galactosidase Source Bovine testes Catalog Number E-BG02 60 µl E-BG µl E-BG µl EC Recommended Reagents included with E-BG02: 400 µl 5x Reaction buffer 500 mm sodium citrate/ phosphate ph 4 Activity 3 U/ml Specific Activity 5 U/mg Optimum ph 4 The supplied buffer concentrate provides the optimal ph for enzyme activity with the standard substrate. If glycosidase treatment is performed at suboptimal ph because of glycoprotein solubility or activity requirements, expect some diminution in enzyme activity. Specific Activity One unit of ß-(1-3,4,6)-Galactosidase is defined as the amount of enzyme required to produce 1 µmole of p-nitrophenol (pnp) in 1 minute at 37 C, ph 4.0 from p-nitrophenyl-ß-d-galactopyranoside. Specificity Cleaves all ß1-3 and ß1-4 linked non-reducing, terminal galactose. ß1-6 linked galactose is released at a slower rate. Formulation The enzyme is provided as a sterile-filtered solution in 20 mm Tris-HCl ph 7.5, 50 mm NaCl, 0.5 mg/ml BSA, ph 7.5. Stability Stable at least 12 months when stored properly. Several days exposure to ambient temperatures will not reduce activity. Storage Store enzyme at -20 C. Purity ß-(1-3,4,6)-Galactosidase is tested for contaminating protease as follows: 10 µg of denatured BSA is incubated at 37 C for 24 hours with 2 µl of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation. Each lot is also tested for contaminating activities by incubating the enzymes with the appropriate substrates for 24 hours; the detection limit is 5 µu/ml (IUB). A passing lot will have no detectable activity. ß-(1-3,4,6)Galactosidase Specifications - Protocol

10 E-BG02 Product Specifications - Protocol Directions for use 1. Add up to 100 µg of asialoglycoprotein or 1 nmol of oligosaccharide to tube. 2. Add deionized water to a total of 14 µl. 3. Add 4 µl of 5x Reaction Buffer Add 2 µl ß-Galactosidase. 5. Incubate at 37 C for 1 hour. For glycoproteins, cleavage may be monitored by SDS- PAGE if the size differential between native and degalactosylated protein is sufficient for detection. Warranties and liabilities, Inc warrants that the above product conforms to the specifications described herein. Should the product fail for reasons other than through misuse will, at its option, replace free of charge or refund the purchase price. This warranty is exclusive and makes no other warrants, expressed or implied, including any implied conditions or warranties of merchantability or fitness for any particular purpose. shall not be liable for any incidental, consequential or contingent damages. This product is intended for in vitro research only. revised on May 28, 2018 References Guile GR, Rudd PM, Wing DR, Prime SB, Dwek RA. A rapid high-resolution high-performance liquid chromatographic method for separating glycan mixtures and analyzing oligosaccharide profiles. Anal Biochem Sep 5;240(2): PMID: Jacob GS, Scudder P. Glycosidases in structural analysis. Methods Enzymol. 1994;230: No abstract available. PMID: Distler JJ, Jourdian GW. The purification and properties of beta-galactosidase from bovine testes. J Biol Chem Oct 10;248(19): PMID:

11 ß(1-4)-Galactosidase Source Recombinant from Streptococcus pnuemonia in E.coli Catalog Number E-BG07 60 µl E-BG µl E-BG µl Recommended Reagents included with E-BG07: 1 vial: Reaction buffer 250mM Sodium phosphate, ph 6.0 Activity 3 U/ml Specific Activity 6 U/mg Molecular Weight ~350,000 dalton ph optimum 6.0, active over the range 5-7. The supplied buffer concentrate provides the optimal ph for enzyme activity with the standard substrate. If glycosidase treatment is performed at suboptimal ph because of glycoprotein solubility or activity requirements, expect some diminution in enzyme activity. Formulation The enzyme is provided as a sterile-filtered solution in 20 mm Tris-HCl, 25 mm NaCl (ph 7.5). Specific Activity One unit of ß-(1-4)-galactosidase is defined as the amount of enzyme required to produce 1 µmole of p-nitrophenol (pnp) in 1 minute at 37 C ph 5 from p-nitrophenyl-beta-d-galactopyranoside. Specificity Non-reducing terminal ß(1-4)-Galactose. Number of antennae does not affect cleavage rate. Fucose linked to the penultimate N-acetylglucosamine will block cleavage of the galactose. Stability Stable at least 12 months when stored properly. Several days exposure to ambient temperatures will not reduce activity. Storage Store enzyme at 4 C. Do not freeze. Purity ß(1-4)-Galactosidase is tested for contaminating protease as follows: 10 µg of denatured BSA is incubated at 37 C for 24 hours with 2 µl of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation. The production host strain has been extensively tested and does not produce any detectable glycosidases. ß-(1-4)Galactosidase Specifications - Protocol

12 E-BG07 Product Specifications - Protocol Directions for use 1. Add up to 100 µg of asialoglycoprotein or 1 nmol of oligosaccharide to tube. 2. Add deionized water to a total of 14 µl. 3. Add 4 µl of 5x Reaction Buffer Add 2 µl ß(1-4) Galactosidase. 5. Incubate at 37 C for 1 hour. For glycoproteins, cleavage may be monitored by SDS- PAGE if the size differential between native and degalactosylated protein is sufficient for detection. Note: The optimum ph for cleavage of oligosaccharides is ~6. References: Glasgow, LR., J.C. Paulson and R.L. Hill. Systematic purification of five glycosidases from Streptococcus pneumoniae. J Biol Chem 252: (1977). Kobata, A. Use of endo- and exoglycosidases for structural studies of glycoconjugates. Anal Biochem 100: 1-14 (1979). Prime, S. J. Dearnley, A.M. Venton, R.B. Parekh and C.J. Edge. Oligosaccharide sequencing based on exo- and endoglycosidase digestion and liquid chromatographic analysis of the products. J Chromatogr A 720: (1996) Dwek, R.A., C.J. Edge, D.J. Harvey, M.R. Wormald and R.B. Parekh. Analysis of glycoprotein-associated oligosaccharides. Ann Rev Biochem 62: (1993) Warranties and liabilities, LLC warrants that the above product conforms to the specifications described herein. Should the product fail for reasons other than through misuse, LLC will, at its option, replace free of charge or refund the purchase price. This warranty is exclusive and, LLC makes no other warrants, expressed or implied, including any implied conditions or warranties of merchantability or fitness for any particular purpose., LLC shall not be liable for any incidental, consequential or contingent damages. This product is intended for in vitro research only. revised on May 29, 2018

13 Endo F1 (Endoglycosidase F1) Endo F1, Endoglycosidase F1, endo-beta-n-acetylglucosaminidase F1 Source recombinant gene from Chryseobacterium meningosepticum in E. Coli Catalog Numbers E-EF01 60 µl E-EF µl E-EF µl EC Recommended Reagents included with E-EF01: 1 vial: 5x Reaction Buffer 250 mm sodium phosphate, ph5.5 Activity 17 U/ml Specific Activity 16 U/mg Molecular Weight 32 kd Specific Activity Defined as the amount of enzyme required to catalyze the release of N-linked oligosaccharides from 1 micromole of denatured Ribonuclease B (RNase B) in 1 minute at 37 C, ph 5.5. Cleavage is monitored by SDS-PAGE (cleaved RNase B migrates faster). Formulation The enzyme is provided as a sterile-filtered solution in 20 mm Tris-HCl, ph 7.5 Stability Several days exposure to ambient temperatures will not reduce activity. Stable at least 12 months when stored properly. Specificity Endo F1 cleaves Asparagine-linked high mannose or hybrid oligosaccharides. It cleaves between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. In contrast, PNGase F removes the oligosaccharide intact. Quality & Purity Endo F1 is tested for contaminating protease as follows: 10 µg of denatured BSA is incubated at 37 C for 24 hours with 2 µl of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation. The production host strain has been extensively tested and does not produce any detectable glycosidases. Storage Store enzyme at 4 C. Do not freeze. Endo F1 Specifications - Protocol

14 Directions for use 1. Add up to 200 µg of glycoprotein to an Eppendorf tube. Adjust to 38 µl final volume with de-ionized water. 2. Add 10 µl 5x Reaction Buffer Add 2.0 µl of Endo F1 to the reaction. Incubate 3 hours at 37 C. Monitor cleavage by SDS-PAGE. References: Maley P., R. B. Trimble, A. L. Tarentino and T. H. Plummer Jr. Characterization of glycoproteins and their associated oligosaccharides through the use of endoglycosidases. Anal Biochem 180: (1989). Plummer, T. H. Jr, A. W. Phelan and A. L. Tarentino. Porcine fibrinogen glycopeptides: substrates for detecting endo-$-n-acetylglucosaminidases F2 and F3. Anal Biochem 235: (1996). Reddy A., B. G. Grimwood, T. H. Plummer Jr and A. L. Tarentino. High-level expression of the Endo-$- Nacetylglucosaminidase F2 gene in E.coli: one step purification to homogeneity. Glycobiology 8: (1998). Tarentino, A. L., C. M. Gomez and T. H. Plummer Jr. Deglycosylation of Asparagine-Linked Glycans by Peptide:N-Glycosidase F. Biochemistry 24: (1985). Tarentino A. L., G. Quinones, W. P. Schrader, L. M. Changchien and T. H. Plummer Jr. Multiple endoglycosidase (Endo) F activities expressed by Flavobacterium meningosepticum. Endo F1: molecular cloning, primary sequence, and structural relationship to Endo H. J Biol Chem 267: (1992). E-EF01 Product Specifications - Protocol specificity of Flavobacterium meningosepticum: Endo F2 and endo F3: purity is the name of the game. Glycobiology 4: (1994). Tarentino, A. L. and T. H. Plummer Jr. Enzymatic deglycosylation of asparagine-linked glycans: purification, properties and specificity of oligosaccharidecleaving enzymes from Flavobacterium meningosepticum. Methods in Enzymology 230:44-57 (1994). Tarentino A. L., G. Quinones and T. H. Plummer Jr. Overexpression and purification of non-glycosylated recombinant endo-$-n-acetylglucosaminidase F3. Glycobiology 5: (1995). Trimble, R. B. and A. L. Tarentino. Identification of Distinct Endoglycosidase (Endo) Activities in Flavobacterium meningosepticum: Endo F1, Endo F2 and Endo F3. J. Biol Chem 266: (1991) Warranties and liabilities warrants that the above product conforms to the specifications described herein. Should the product fail for reasons other than through misuse will, at its option, replace free of charge or refund the purchase price. This warranty is exclusive and makes no other warrants, expressed or implied, including any implied conditions or warranties of merchantability or fitness for any particular purpose. shall not be liable for any incidental, consequential or contingent damages. This product is intended for in vitro research only. revised on May 28, 2018 Tarentino A. L., G. Quinones, L. M. Changchien, and T. H. Plummer Jr. Multiple endoglycosidase F activities expressed by Flavobacterium meningosepticum endoglycosidases F2 and F3: Molecular cloning, primary sequence, and enzyme expression. J Biol Chem 268(13): (1993). Tarentino A. L. and T. H. Plummer Jr. Substrate

15 Endo F2 (Endoglycosidase F2) Endo-beta-N-acetylglucosaminidase F2 Source recombinant gene from Chryseobacterium meningosepticum in E. Coli Catalog Number E-EF02 E-EF02 60 µl E-EF µl E-EF µl EC Recommended Reagents included with E-EF02: 1 vial: 5x Reaction Buffer µl 250 mm sodium acetate, ph4.5 Activity 5 U/ml Specific Activity 20 U/mg Molecular Weight 32 kd Specific Activity Defined as the amount of enzyme required to catalyze the release of N-linked oligosaccharides from 1 micromole of denatured Ribonuclease B (RNase B) in 1 minute at 37 C, ph 5.5. Cleavage is monitored by SDS-PAGE (cleaved RNase B migrates faster). Formulation The enzyme is provided as a sterile-filtered solution in 10 mm sodium acetate, 25mM NaCl, ph 4.5 Storage Store enzyme at 4 C. Do not freeze. Specificity Endo F2 cleaves Asparagine-linked high mannose or biantennary oligosaccharides. It cleaves between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. In contrast, PNGase F removes the oligosaccharide intact. Quality & Purity Endo F2 is tested for contaminating protease as follows: 10 µg of denatured BSA is incubated at 37 C for 24 hours with 2 µl of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation. The production host strain has been extensively tested and does not produce any detectable glycosidases. Stability Several days exposure to ambient temperatures will not reduce activity. Stable at least 12 months when stored properly. Endo F2 Specifications - Protocol

16 E-EF02 Product Specifications - Protocol Directions for use 1. Add up to 200 µg of glycoprotein to an Eppendorf tube. Adjust to 38 µl final volume with de-ionized water. 2. Add 10 µl 5x Reaction Buffer Add 2.0 µl of Endo F2 to the reaction. Incubate 3 hours at 37 C. Monitor cleavage by SDS-PAGE. References: Maley P., R. B. Trimble, A. L. Tarentino and T. H. Plummer Jr. Characterization of glycoproteins and their associated oligosaccharides through the use of endoglycosidases. Anal Biochem 180: (1989). Plummer, T. H. Jr, A. W. Phelan and A. L. Tarentino. Porcine fibrinogen glycopeptides: substrates for detecting endo-$-n-acetylglucosaminidases F2 and F3. Anal Biochem 235: (1996). Reddy A., B. G. Grimwood, T. H. Plummer Jr and A. L. Tarentino. High-level expression of the Endo-$- Nacetylglucosaminidase F2 gene in E.coli: one step purification to homogeneity. Glycobiology 8: (1998). Tarentino, A. L., C. M. Gomez and T. H. Plummer Jr. Deglycosylation of Asparagine-Linked Glycans by Peptide:N-Glycosidase F. Biochemistry 24: (1985). Tarentino A. L., G. Quinones, W. P. Schrader, L. M. Changchien and T. H. Plummer Jr. Multiple endoglycosidase (Endo) F activities expressed by Flavobacterium meningosepticum. Endo F1: molecular cloning, primary sequence, and structural relationship to Endo H. J Biol Chem 267: (1992). Tarentino A. L. and T. H. Plummer Jr. Substrate specificity of Flavobacterium meningosepticum: Endo F2 and endo F3: purity is the name of the game. Glycobiology 4: (1994). Tarentino, A. L. and T. H. Plummer Jr. Enzymatic deglycosylation of asparagine-linked glycans: purification, properties and specificity of oligosaccharidecleaving enzymes from Flavobacterium meningosepticum. Methods in Enzymology 230:44-57 (1994). Tarentino A. L., G. Quinones and T. H. Plummer Jr. Overexpression and purification of non-glycosylated recombinant endo-$-n-acetylglucosaminidase F3. Glycobiology 5: (1995). Trimble, R. B. and A. L. Tarentino. Identification of Distinct Endoglycosidase (Endo) Activities in Flavobacterium meningosepticum: Endo F1, Endo F2 and Endo F3. J. Biol Chem 266: (1991). Warranties and liabilities warrants that the above product conforms to the specifications described herein. Should the product fail for reasons other than through misuse will, at its option, replace free of charge or refund the purchase price. This warranty is exclusive and makes no other warrants, expressed or implied, including any implied conditions or warranties of merchantability or fitness for any particular purpose. shall not be liable for any incidental, consequential or contingent damages. This product is intended for in vitro research only. revised on May 28, 2018 Tarentino A. L., G. Quinones, L. M. Changchien, and T. H. Plummer Jr. Multiple endoglycosidase F activities expressed by Flavobacterium meningosepticum endoglycosidases F2 and F3: Molecular cloning, primary sequence, and enzyme expression. J Biol Chem 268(13): (1993) phone/fax info@.com phone fax

17 Endo F3 (Endoglycosidase F3) Endo-beta-N-acetylglucosaminidase F3 Source recombinant gene from Chryseobacterium meningosepticum in E. Coli Catalog Number E-EF03 60 µl E-EF µl E-EF µl EC Recommended Reagents included with E-EF03: 1 vial: 5x Reaction Buffer µl 250 mm sodium acetate, ph4.5 Activity 5 U/ml Specific Activity 25 U/mg Molecular Weight 30 kd Specific Activity Defined as the amount of enzyme required to catalyze the release of N-linked oligosaccharides from 1 micromole of porcine fibrininogen in 1 minute at 37 C, ph 4.5. Cleavage is monitored by SDS-PAGE. Formulation The enzyme is provided as a sterile-filtered solution in 20 mm Tris-HCl, ph 7.5 Specificity Endo F3 cleaves free or Asparagine-linked triantennary or fucosylated biantennary oligosaccharides,as well as triamnnosyl chitobiose core structures. It cleaves between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. In contrast, PNGase F removes the oligosaccharide intact. Alpha 1-3 fucosylation will inhibit enzymatc activity. The recombinant version is not glycosylated, which may result in properties differing from the native protein. Quality & Purity Endo F3 is tested for contaminating protease as follows: 10 µg of denatured BSA is incubated at 37 C for 24 hours with 2 µl of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation. The production host strain has been extensively tested and does not produce any detectable glycosidases. Stability Several days exposure to ambient temperatures will not reduce activity. Stable at least 12 months when stored properly. Storage Store enzyme at 4 C. Do not freeze. Endo F3 Specifications - Protocol

18 E-EF03 Product Specifications - Protocol Directions for use 1. Add up to 200 µg of glycoprotein to an Eppendorf tube. Adjust to 38 µl final volume with de-ionized water. 2. Add 10 µl 5x Reaction Buffer Add 2.0 µl of Endo F3 to the reaction. Incubate 3 hours at 37 C. Monitor cleavage by SDS-PAGE. References: Maley P., R. B. Trimble, A. L. Tarentino and T. H. Plummer Jr. Characterization of glycoproteins and their associated oligosaccharides through the use of endoglycosidases. Anal Biochem 180: (1989). Plummer, T. H. Jr, A. W. Phelan and A. L. Tarentino. Porcine fibrinogen glycopeptides: substrates for detecting endo-$-n-acetylglucosaminidases F2 and F3. Anal Biochem 235: (1996). Reddy A., B. G. Grimwood, T. H. Plummer Jr and A. L. Tarentino. High-level expression of the Endo-$- Nacetylglucosaminidase F2 gene in E.coli: one step purification to homogeneity. Glycobiology 8: (1998). Tarentino, A. L., C. M. Gomez and T. H. Plummer Jr. Deglycosylation of Asparagine-Linked Glycans by Peptide:N-Glycosidase F. Biochemistry 24: (1985). Tarentino A. L., G. Quinones, W. P. Schrader, L. M. Changchien and T. H. Plummer Jr. Multiple endoglycosidase (Endo) F activities expressed by Flavobacterium meningosepticum. Endo F1: molecular cloning, primary sequence, and structural relationship to Endo H. J Biol Chem 267: (1992). Tarentino A. L. and T. H. Plummer Jr. Substrate specificity of Flavobacterium meningosepticum: Endo F2 and endo F3: purity is the name of the game. Glycobiology 4: (1994). Tarentino, A. L. and T. H. Plummer Jr. Enzymatic deglycosylation of asparagine-linked glycans: purification, properties and specificity of oligosaccharidecleaving enzymes from Flavobacterium meningosepticum. Methods in Enzymology 230:44-57 (1994). Tarentino A. L., G. Quinones and T. H. Plummer Jr. Overexpression and purification of non-glycosylated recombinant endo-$-n-acetylglucosaminidase F3. Glycobiology 5: (1995). Trimble, R. B. and A. L. Tarentino. Identification of Distinct Endoglycosidase (Endo) Activities in Flavobacterium meningosepticum: Endo F1, Endo F2 and Endo F3. J. Biol Chem 266: (1991) Warranties and liabilities warrants that the above product conforms to the specifications described herein. Should the product fail for reasons other than through misuse will, at its option, replace free of charge or refund the purchase price. This warranty is exclusive and makes no other warrants, expressed or implied, including any implied conditions or warranties of merchantability or fitness for any particular purpose. shall not be liable for any incidental, consequential or contingent damages. This product is intended for in vitro research only. revised on May 28, 2018 Tarentino A. L., G. Quinones, L. M. Changchien, and T. H. Plummer Jr. Multiple endoglycosidase F activities expressed by Flavobacterium meningosepticum endoglycosidases F2 and F3: Molecular cloning, primary sequence, and enzyme expression. J Biol Chem 268(13): (1993).

19 Endo H (Endoglycosidase H) endo-beta-n-acetylglucosaminidase H Source recombinant gene from Streptomyces plicatus in E. Coli Catalog Number E-EH02 60 µl E-EH µl E-EH µl EC Recommended Reagents included with E-EH02: 1 vial: 5x Reaction Buffer ml 250 mm sodium phosphate, ph5.5 1 vial: Denaturation Solution ml 2% SDS, 1 M Beta-mercaptoethanol Activity 5 U/ml Specific Activity 40 U/mg Applications Releases asparagine-linked hybrid or high mannose oligosaccharides, but not complex oligosaccharides. Endo H cleaves between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. In contrast, PNGase F removes the oligosaccharide intact. Detergent and heat denaturation may increase the rate of cleavage for some glycoproteins. Specific Activity One unit of Endo H activity is defined as the amount of enzyme required to catalyze the release of N-linked oligosaccharides from 1 µmole of denatured Ribonuclease B. Cleavage is monitored by SDS-PAGE (cleaved Ribonuclease B migrates faster). Specificity Endo H cleaves Asparagine-linked hybrid or high mannose oligosaccharides, but not complex oligosaccharides. It cleaves between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. In contrast, PNGase F removes the oligosaccharide intact. Detergent and heat denaturation may increase the rate of cleavage for some glycoproteins. Formulation The enzyme is provided as a sterile-filtered solution in 20 mm Tris-HCl, 25mM NaCl, 1 mm EDTA (ph 7.5). Molecular Weight approximately 29 kd. ph optimum: 5.5, active over the range 5-6. Storage Store enzyme at 4 C. Do not freeze. Stability Several days exposure to ambient temperatures will not reduce activity. Stable at least 12 months when stored properly. Endo H Specifications - Protocol

20 E-EH02 Product Specifications - Protocol Quality & Purity Endo H is tested for contaminating protease as follows: 10 µg of denatured BSA is incubated at 37 C for 24 hours with 2 µl of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation. The production host strain has been extensively tested and does not produce any detectable glycosidases. Directions for use 1. Add up to 200 µg of glycoprotein to an Eppendorf tube. Adjust to 37.5 µl final volume with de-ionized water. 2. Add 10 µl 5x Reaction Buffer 5.5 and 2.5 µl of Denaturation Solution. Heat at 100 C for 5 minutes. NOTE: It is not necessary to add Triton X-100. SDS will not inactivate Endo H. 4. Add 2.0 µl of Endo H to the reaction. Incubate 3 hours at 37 C. If SDS or heat denaturation is omitted, increase incubation time to at least 24 hours. Monitor cleavage by SDS-PAGE. References: Robbins P. W., D. F. Wirth and C. J. Hering. Expression of the Streptomyces enzyme endoglycosidase H in Escherichia coli. Biol Chem 256: (1981). Robbins P. W., R. B. Trimble, D. F. Wirth, C. Hering, F. Maley, G. F. Maley, R. Das, B. W. Gibson, N. Royal and K. Biemann. Primary structure of the Streptomyces enzyme endo-beta-n-acetylglucosaminidase H. J Biol Chem 259: (1984). Trimble R. B., A. L. Tarentino, G. E Aumick and F. Maley. Endo-beta-N-acetylglucosaminidase L from Streptomyces plicatus. Methods Enzymol 83: (1982). Trimble R. B. and F. Maley. Optimizing hydrolysis of N-linked high-mannose oligosaccharides by endo-beta- Nacetylglucosaminidase H. Anal Biochem 141: (1984). Trimble R. B., R. J. Trumbly and F. Maley. Endo-beta- Nacetylglucosaminidase H from Streptomyces plicatus. Methods Enzymol 138: (1987). Trumbly R. J., P. W. Robbins, M. Belfort, F. D. Ziegler, F. Maley and R. B. Trimble. Amplified expression of Streptomyces endo-beta-n-acetylglucosaminidase H in Escherichia coli and characterization of the enzyme product. J Biol Chem 260: (1985). Warranties and liabilities warrants that the above product conforms to the specifications described herein. Should the product fail for reasons other than through misuse will, at its option, replace free of charge or refund the purchase price. This warranty is exclusive and makes no other warrants, expressed or implied, including any implied conditions or warranties of merchantability or fitness for any particular purpose. shall not be liable for any incidental, consequential or contingent damages. This product is intended for in vitro research only. revised on May 28, 2018

21 α-(1-6) Fucosidase α-l-fucoside fucohydralase Source recombinant Elizabethkingia miricola in E. Coli Catalog Number E-F µl E-F µl E-F µl EC Recommended Reagents included with E-F006: 1 vial: 5x Reaction buffer 250 mm NaHPO 4, ph 5 Activity 1 U/ml Specific Activity 1.8 U/mg Molecular Weight ~50,000 daltons Specific Activity One unit of α-(1-6) Fucosidase is defined as the amount of enzyme required to produce 1 µmole of methylumbelliferone in 1 minute at 37 C, ph 5.0 from 4-methylumbelliferyl-α-L-fucopyranoside. Formulation The enzyme is provided as a sterile-filtered solution in 20 mm Tris HCl ph 7.5 and 25 mm NaCl. Storage Store enzyme at 4 C. Do not freeze. Specificity α(1-6) linked core fucose when covalently attached to a reporter molecule at the reducing terminus. The one exception is that a terminal unbranched α(1-3) ) or α(1-4) fucose is cleaved in the absence of any reporter molecule. These substrates do not apparently occur in nature. Reporter molecules known to support cleavage are amino-napthalene disulfonic and trisulfonic acids and 2-aminobenzoic acid(2-aa). However, 2-aminobenzamide(2-AB) will not support cleavage. Shorter oligosaccharides such as trimannosylchitobiose are more completely digested than longer derivatives which may require longer incubation times. Stability Stable at least 12 months when stored properly. Several days exposure to ambient temperatures will not reduce activity. Purity α-(1-6) Fucosidase is tested for contaminating protease as follows: 10 µg of denatured BSA is incubated at 37 C for 24 hours with 2 µl of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation. Each lot is also tested for contaminating activities by incubating the enzymes with the appropriate substrates for 24 hours; the detection limit is 5 µu/ml (IUB). A passing lot will have no detectable activity. α-(1-6) Fucosidase Specifications - Protocol

22 E-F006 Product Specifications - Protocol Directions for use 1. Add up to 1 nmol of labeled oligosaccharide to tube. 2. Add de-ionized water to a total of 15 µl. 3. Add 4 µl 5x Reaction Buffer Add 1 µl α-(1-6) Fucosidase. 5. Incubate overnight at 37 C. Warranties and liabilities, LLC warrants that the above product conforms to the specifications described herein. Should the product fail for reasons other than through misuse, LLC will, at its option, replace free of charge or refund the purchase price. This warranty is exclusive and, LLC makes no other warrants, expressed or implied, including any implied conditions or warranties of merchantability or fitness for any particular purpose., LLC shall not be liable for any incidental, consequential or contingent damages. This product is intended for in vitro research only. revised on May 28, 2018

23 α-(1-3,4) Fucosidase α-l-fucoside fucohydralase Source Xanthamonas manihotis Catalog Numbers E-F µl E-F µl E-F µl EC Recommended Reagents included with E-F134: 1 vial: 5x Reaction buffer 250 mm NaHPO 4, ph 5 Activity 0.5 U/ml Specific Activity 2 U/mg Application Deglycosylating of proteins with Lewis structures Molecular Weight ~62,000 daltons Specific Activity One unit of Fucosidase is defined as the amount of enzyme required to cleave 1 µmole of fucose from Lewis X trisaccharide, 4-methylumbelliferyl glycoside in 1 minute at 37 C, ph 5.0. Specificity α-(1-3,4)-fucosidase cleaves α-(1-3) and α-(1-4) -linked fucose GlcNAc of a Gal-GlcNAc disaccharide structure. The presence of sialic acid (but not fucose) linked to the galactose will block cleavage. Formulation The enzyme is provided as a sterile-filtered solution in 20 mm Tris HCl ph 7.5 and 25 mm NaCl. Stability Stable at least 12 months when stored properly. Several days exposure to ambient temperatures will not reduce activity. Storage Store enzyme at 4 C. Do not freeze. Purity α-(1-3,4) Fucosidase is tested for contaminating protease as follows: 10 µg of denatured BSA is incubated at 37 C for 24 hours with 2 µl of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation. Each lot is also tested for contaminating activities by incubating the enzymes with the appropriate substrates for 24 hours; the detection limit is 5 µu/ml (IUB). A passing lot will have no detectable activity. α-(1-3,4) Fucosidase Specifications - Protocol

24 E-F134 Product Specifications - Protocol Directions for use 1. Add up to 1 nmol of oligosaccharide to tube. 2. Add de-ionized water to a total of 15 µl. 3. Add 4 µl 5x Reaction Buffer Add 1 µl α-(1-3,4) Fucosidase. 5. Incubate 1 hour at 37 C. Progress may be monitored by SDS-PAGE if the size differential between native and de-glycosylated protein is sufficient for detection. Warranties and liabilities, Inc warrants that the above product conforms to the specifications described herein. Should the product fail for reasons other than through misuse, Inc will, at its option, replace free of charge or refund the purchase price. This warranty is exclusive and, Inc makes no other warrants, expressed or implied, including any implied conditions or warranties of merchantability or fitness for any particular purpose., Inc shall not be liable for any incidental, consequential or contingent damages. This product is intended for in vitro research only. revised on May 28, 2018

25 O-Glycosidase Endo-alpha-N-Acetylgalactosaminidase Source recombinant Streptococcus pneumoniae in E.Coli EC Catalog Number E-G µl E-G µl E-G µl Recommended Reagents included with E-G001: 1 vial: 5x Reaction buffer 250 mm sodium phosphate, ph 5.0 Activity 1.25 U/ml Specific Activity 12 U/mg Specific Activity One unit of O-Glycosidase is defined as the amount of enzyme required to produce 1 µmole of p-nitrophenol (pnp) in 1 minute at 37 C, ph 5.0 from p-nitrophenyl- 2-acetamido-2-deoxy-3-O-(beta-D-galactopyranosyl)- alpha-d-galactopyranoside. Storage Store enzyme at 4 C. Do not freeze. Formulation The enzyme is provided as a sterile-filtered solution in 50 mm sodium phosphate (ph 7.5). Molecular Weight ~180,000 daltons ph Optimum 5, active over the range 5-7. Specifictity Cleaves only unsubstituted Gal-ß(1-3)GalNAcalpha disaccharides attached to the serine or threonine residues of glycoproteins or glycopeptides. Substitutions such as sialic acid, galactose, fucose or N-acetylglucosamine must first be removed with the appropriate exoglycosidase prior to treatment with O-Glycosidase. At minimum, a sialadase such as Sialidase Au (Alpha-2-3,6,8,9), part number E-S001, is almost always required to remove sialic acids Purity O-Glycosidase is tested for contaminating protease as follows: 10 µg of denatured BSA is incubated at 37 C for 24 hours with 2 µl of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation. The production host strain has been extensively tested and does not produce any detectable glycosidases. O-Glycosidase Specifications - Protocol

26 Directions for use 1. Add up to 100 µg of glycoprotein to tube. 2. Add de-ionized water to a total of 13 µl. 3. Add 4 µl 5x Reaction Buffer Add 1 µl Sialidase AU (E-S001) 5. Add 2 µl O-Glycosidase. 6. Incubate at 37 C for 1 hour. Cleavage may be monitored by SDS-PAGE if the size differential between native and de-o-glycosylated protein is sufficient for detection. E-G001 Product Specifications - Protocol Warranties and liabilities warrants that the above product conforms to the attached analytical documents. Should the product fail for reasons other than through misuse will, at its option, replace free of charge or refund the purchase price. This warranty is exclusive and makes no other warrants, expressed or implied, including any implied conditions or warranties of merchantability or fitness for any particular purpose. shall not be liable for any incidental, consequential or contingent damages. This product is intended for in vitro research only. revised on May 28, 2018 References Bhavanandan, V.P., J. Umemoto and E.A. Davidson. Characterization of an endo-alpha-nacetylgalactosaminidase from Diplococcus pneumoniae. Biochem Biophys 70: (1976). Fan, J. Q., K. Yamamoto, H. Kumagai and T. Tochikura. Induction and efficient purification of endo-alpha-nacetyl-d-galactosaminidase from Alcaligenes sp. Agric Biol Chem 54: (1990). Glasgow, L R., J. C. Paulson and R. L. Hill. Systematic purification of five glycosidases from Streptococcus pneumoniae. J Biol Chem 252: (1977). Iwase, H. and K. Hotta. Release of O-linked glycoprotein glycans by endo-alpha-n-acetyl-d-galactosaminidase. Methods Mol Biol 14: (1993). Unemoto, J., V. P. Bhavanandan and E. A. Davidson. Purification and properties of an endo-alpha-n-acetyl- D-galactosaminidase from Diplococcus pneumoniae. J Biol Chem 252: (1977).

27 ß-N-acetylglucosaminidase Glucosaminidase Source recombinant gene from Streptococcus pneumoniae in E. Coli Catalog Number E-GL01 60 µl E-GL µl E-GL µl EC Recommended Reagents included with E-GL01: 1 vial: 5x Reaction buffer 250mM Sodium phosphate, ph 5 Activity 40 U/ml Specific Activity 80 U/mg Application Structural analysis of oligosaccharides Distinguishing different N-acetyl glucosamine linkages Distinguishing between N-acetyl glucosamine and N- acetylgalactosamine Removing heterogeneity from glycoproteins Molecular Weight ~140,000 daltons ph optimum 5.0, active over the range 5-7 Specific Activity One unit of ß-N-acetylglucosaminidase is defined as the amount of enzyme required to produce 1 µmole of p-nitrophenol (pnp) in 1 minute at 37 C, ph 5.0 from p-nitrophenyl-ß-d-n-acetyl-glucosaminide. Specificity All non-reducing terminal ß-linked N-acetylglucosamine. Bisecting GlcNAc slows the reaction. Formulation The enzyme is provided as a sterile-filtered solution in 20 mm Tris-HCl, 25 mm NaCl (ph 7.5). Stability Stable at least 12 months when stored properly. Several days exposure to ambient temperatures will not reduce activity. Storage Store enzyme at 4 C. Do not freeze. Purity ß-N-acetylglucosaminidase is tested for contaminating protease as follows: 10 µg of denatured BSA is incubated at 37 C for 24 hours with 2 µl of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation. The production host strain has been extensively tested and does not produce any detectable glycosidases. ß-N-Acetylglucosaminidase Specifications - Protocol

28 E-GL01 Product Specifications - Protocol Directions for use 1. Add up to 100 µg of asialogalacto-glycoprotein or 1 nmol of oligosaccharide to tube. 2. Add de-ionized water to a total of 14 µl. 3. Add 4 µl 5x Reaction Buffer Add 2 µl ß-N-acetylglucosaminidase 5. Incubate at 37 C for 3 hours. If bisecting GlcNAc is present, incubation time should be increased to 12 hours. Progress may be monitored by SDS-PAGE if the size differential between native and de-glycosylated protein is sufficient for detection. References Clarke, V. A., N. Platt and T.D. Betters. Cloning and expression of the beta-n-acetylglucosaminidase gene from Steptococcus pneumoniae. Generation of truncated enzymes with modified aglyconn specificity. J Biol Chem 270: (1995). Dwek, R. A., C. J. Edge, D. J. Harvey, M. R. Wormald and R. B. Parekh. Analysis of glycoprotein-associated oligosaccharides. Ann Rev Biochem 62: (1993). Glasgow, L.R., J.C. Paulson and R.L. Hill. Systematic purification of five glycosidases from Streptococcus pneumoniae. J Biol Chem 252: (1977). Kobata, A. Use of endo- and exoglycosidases for structural studies of glycoconjugates. Anal Biochem 100: 1-14 (1979). Prime, S., J. Dearnley, A. M. Venton, R. B. Parekh and C. J. Edge. Oligosaccharide sequencing based on exo- and endoglycosidase digestion and liquid chromatographic analysis of the products. J Chromatogr A 720: (1996). Warranties and liabilities Inc warrants that the above product conforms to the specifications described herein. Should the product fail for reasons other than through misuse Inc will, at its option, replace free of charge or refund the purchase price. This warranty is exclusive and Inc makes no other warrants, expressed or implied, including any implied conditions or warranties of merchantability or fitness for any particular purpose. Inc shall not be liable for any incidental, consequential or contingent damages. This product is intended for in vitro research only. revised on May 28, 2018

29 PNGase F (Peptide-N-Glycosidase F) Peptide-N4-(acetyl-ß-glucosaminyl)-asparagine amidase N-Glycosidase F Source Elizabethkingia meningosepticum was (Chyrseobacterium/Flavobacterium men.) Catalog Numbers E-PNG01 60 µl 0.3 U E-PNG µl 0.1 U E-PNG µl 1.0 U EC Recommended Reagents included with E-PNG01: 1 vial: 5x Reaction Buffer ph µl 1 vial: Denaturation Solution µl 2% SDS/ 1 M β-mercaptoethanol 1 vial: 15% Triton X µl Activity 5 U/ml Specific Activity 25 U/mg Molecular Weight approximately 35 kd. ph optimum: 7.5, active over the range Storage Store enzyme at 4 C. Do not freeze. Specific Activity One unit of PNGase F activity is defined as the amount of enzyme required to catalyze the release of N-linked oligosaccharides from 1 micromole of RNase B in 1 minute at 37 C, ph 7.5. Cleavage is monitored by SDS-PAGE (cleaved RNase B migrates faster). Formulation The enzyme is provided as a sterile-filtered solution in 20 mm Tris-HCl (ph 7.5). Specificity PNGase F cleaves asparagine-linked (N-linked) oligosaccharides from glycoproteins. PNGase F deaminates asparagine to aspartic acid, leaving the oligosaccharides intact. Denaturation increases the rate of cleavage up to 100x. Most native proteins can still be completely N-deglycosylated but incubation time must be increased. PNGase F will remain active under incubation conditions for at least 72 hours. PNGase F will not remove oligosaccharides containing Alpha-(1,3)-linked core fucose commonly found on plant glycoproteins; for this purpose, use peptide N-glycosidase A. Stability Several days exposure to ambient temperatures will not reduce activity. Stable at least 12 months when stored properly. Quality & Purity PNGase F is tested for contaminating protease as follows: 10 µg of denatured BSA is incubated at 37 C for 24 hours with 2 µl of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation. continued PNGase F Specifications - Protocol

30 PNGase F - Product Specifications & Protocol Quality & Purity continued The absence of exoglycosidase contaminants is confirmed by extended incubations with the corresponding pnpglycosides. PNGase F is isolated from culture supernatants of Elizabethkingia (Chryseobacterium or Flavobacterium) meningosepticum. Significant contaminants are the endoglycosidase F enzymes, which cleave within the diacetylchitobiose core of some N-linked oligosaccharides leaving an N-acetylglucosamine residue attached to the asparagine. These contaminants are chromatographically removed from PNGase F preparations. Directions for use 1. Add up to 200µg of glycoprotein to an Eppendorf tube. Adjust to 35 µl final volume with de-ionized water. 2. Add 10 µl 5x Reaction Buffer 7.5 and 2.5 µl of Denaturation Solution. Heat at 100 C for 5 minutes. 3. Cool. Add 2.5 µl of Triton X-100 and mix. NOTE: Failure to add Triton X-100 will result in a 3-fold reduction of PNGase F activity. 4. Add 2.0 µl of PNGase F to the reaction. Incubate 3 hours at 37 C. If SDS or heat denaturation is omitted, increase incubation time to at least 24 hours. Monitor cleavage by SDS-PAGE. References: Bayer, E.A., F. De Meester, T. Kulik and M. Wilchek. Preparation of deglycosylated egg white avidin. Appl Biochem Biotech 53: 1-9 (1995) Elder, J.H. and S. Alexander. endo-b-n-acetylglucosaminidase F: endoglycosidase from Flavobacterium meningosepticum that cleaves both high-mannose and complex glycoproteins. Proc Natl Acad Sci USA 79: (1982) Tarentino, A.L., C.M. Gomez an d T.H. Plummer, Jr. Deglycosylation of asparagine-linked glycans by peptide :N-glycosidase F. Biochemistry 24: (1985) Tarentino A.L. and T.H. Plummer. Enzymatic deglycosylation of asparagine-linked glycans: purification, properties, and specificity of oligosaccharide-cleaving enzymes from Flavobacterium meningosepticum. Meth Enzymol 230:44-57 (1994) Trimble R.B. and A.L. Tarentino. Identification of distinct endoglycosidase (endo) activities in Flavobacterium meningosepticum: endo F1, endo F2 and endo F3. Endo F1 and endo H hydrolyze only high mannose and hybrid glycans. J Biol Chem 266: (1991). Taga, E. M., A. Waheed and R. L. Van Etten. Structural and chemical characterization of a homogeneous peptide N-glycosidase from almond. Biochemistry 23: (1984). Warranties and liabilities Inc warrants that the above product conforms to the specifications described herein. Should the product fail for reasons other than through misuse will, at its option, replace free of charge or refund the purchase price. This warranty is exclusive and makes no other warrants, expressed or implied, including any implied conditions or warranties of merchantability or fitness for any particular purpose. shall not be liable for any incidental, consequential or contingent damages. This product is intended for in vitro research only. revised May 28, 2018

31 PNGase F (Peptide-N-Glycosidase F) Peptide-N4-(acetyl-ß-glucosaminyl)-asparagine amidase N-Glycosidase F Source recombinant from Elizabethkingia meningosepticum was (Chyrseobacterium/Flavobacterium men.) Catalog Number E-RPNG01 EC Specificity PNGase F cleaves asparagine-linked (N-linked) oligosaccharides from glycoproteins. PNGase F deaminates asparagine to aspartic acid, leaving the oligosaccharides intact. Applications Amino acid sequence determination X-Ray crystallography Removing heterogeneity due to carbohydrates Studying carbohydrate ligand binding Removing carbohydrate epitopes from antigens Studying the role of glycosylation in protein folding and activity. Contents 1 vial: PNGase F - 60 µl (0.3 U) 1 vial: 5x Reaction Buffer ph µl 1 vial: Denaturation Solution µl 2% SDS/ 1 M β-mercaptoethanol 1 vial: 15% Triton X µl Specific Activity 25 U/mg Activity 5 U/ml Specific Activity One unit of PNGase F activity is defined as the amount of enzyme required to catalyze the release of N-linked oligosaccharides from 1 micromole of RNase B in 1 minute at 37 C, ph 7.5. Cleavage is monitored by SDS-PAGE (cleaved RNase B migrates faster). Denaturation increases the rate of cleavage up to 100x. Most native proteins can still be completely N-deglycosylated but incubation time must be increased. PNGase F will remain active under incubation conditions for at least 72 hours. PNGase F will not remove oligosaccharides containing Alpha-(1,3)-linked core fucose commonly found on plant glycoproteins; for this purpose, use peptide N-glycosidase A. Formulation The enzyme is provided as a sterile-filtered solution in 20 mm Tris-HCl (ph 7.5). Molecular Weight approximately 35 kd. ph optimum: 7.5, active over the range Storage Store enzyme at 4 C. Do not freeze. Stability Several days exposure to ambient temperatures will not reduce activity. Stable at least 12 months when stored properly. E-RPNG01 PNGase F Specifications - Protocol

32 E-RPNG01 Product Specifications - Protocol Quality & Purity PNGase F is tested for contaminating protease as follows: 10 µg of denatured BSA is incubated at 37 C for 24 hours with 2 µl of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation. The production host strain has been extensively tested and does not produce any detectable glycosidases. Directions for use 1. Add up to 200µg of glycoprotein to an Eppendorf tube. Adjust to 35 µl final volume with de-ionized water. 2. Add 10 µl 5x Reaction Buffer 7.5 and 2.5 µl of Denaturation Solution. Heat at 100 C for 5 minutes. 3. Cool. Add 2.5 µl of Triton X-100 and mix. NOTE: Failure to add Triton X-100 will result in a 3-fold reduction of PNGase F activity. 4. Add 2.0 µl of PNGase F to the reaction. Incubate 3 hours at 37 C. If SDS or heat denaturation is omitted, increase incubation time to at least 24 hours. Monitor cleavage by SDS-PAGE. References: Bayer, E.A., F. De Meester, T. Kulik and M. Wilchek. Preparation of deglycosylated egg white avidin. Appl Biochem Biotech 53: 1-9 (1995) Elder, J.H. and S. Alexander. endo-b-n-acetylglucosaminidase F: endoglycosidase from Flavobacterium meningosepticum that cleaves both high-mannose and complex glycoproteins. Proc Natl Acad Sci USA 79: (1982) Tarentino, A.L., C.M. Gomez an d T.H. Plummer, Jr. Deglycosylation of asparagine-linked glycans by peptide :N-glycosidase F. Biochemistry 24: (1985) Tarentino A.L. and T.H. Plummer. Enzymatic deglycosylation of asparagine-linked glycans: purification, properties, and specificity of oligosaccharide-cleaving enzymes from Flavobacterium meningosepticum. Meth Enzymol 230:44-57 (1994) Trimble R.B. and A.L. Tarentino. Identification of distinct endoglycosidase (endo) activities in Flavobacterium meningosepticum: endo F1, endo F2 and endo F3. Endo F1 and endo H hydrolyze only high mannose and hybrid glycans. J Biol Chem 266: (1991). Taga, E. M., A. Waheed and R. L. Van Etten. Structural and chemical characterization of a homogeneous peptide N-glycosidase from almond. Biochemistry 23: (1984). Warranties and liabilities, Inc warrants that the above product conforms to the specifications described herein. Should the product fail for reasons other than through misuse will, at its option, replace free of charge or refund the purchase price. This warranty is exclusive and makes no other warrants, expressed or implied, including any implied conditions or warranties of merchantability or fitness for any particular purpose. shall not be liable for any incidental, consequential or contingent damages. This product is intended for in vitro research only. revised on May 24, 2018

33 Sialidase Au Alpha-(2-3,6,8,9) Nueraminidase, NANase Source recombinant from Arthrobacter ureafaciens in E. Coli Catalog Number E-S µl E-S µl E-S µl EC Applications Structural analysis of oligosaccharides Determining sialic acid linkage Glycoprotein deglycosylation Removing heterogeneity from glycoproteins Recommended Reagents included with E-S001: 1 vial: Reaction buffer 250mM Sodium phosphate, ph 6.0 Activity 5 U/m Specific Activity 135 U/mgl Molecular Weight ~69,000 daltons ph optimum 6.0, active over the range Specific Activity One unit of Sialidase Au is defined as the amount of enzyme required to produce 1 µmole of methylumbelliferone in 1 minute at 37 C, ph 5.0 from MU-NANA (2.-(4-methyl-umbelliferyl)-alpha-D-N acetylneuraminic acid]. Specificity All non-reducing terminal branched and unbranched sialic acid. Formulation The enzyme is provided as a sterile-filtered solution in 20 mm Tris-HCl, 25 mm NaCl (ph 7.5). Stability Stable at least 12 months when stored properly. Several days exposure to ambient temperatures will not reduce activity. Storage Store enzyme at 4 C. Do not freeze. Purity Sialidase Au is tested for contaminating protease as follows: 10 µg of denatured BSA is incubated at 37 C for 24 hours with 2 µl of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation. 50 mm sodium phosphate (ph 6.0) provides the optimal buffer for enzyme activity with sialyllactose, a standard substrate. If glycosidase treatment is performed at suboptimal ph because of glycoprotein solubility or activity requirements, expect some diminution in enzyme activity. The production host strain has been extensively tested and does not produce any detectable glycosidases. Sialidase Au Specifications - Protocol

34 E-S001 Product Specifications - Protocol Directions for use 1. Add up to 100 µg of glycoprotein or 1 nmol of oligosaccharide to tube. 2. Add de-ionized water to a total of 14 µl. 3. Add 4 µl Reaction Buffer Add 2 µl Sialidase Au. 5. Incubate at 37 C for 1 hour. NOTE: longer incubation times are necessary if branched sialic acids are present. Desialylation may be monitored by SDS-PAGE if the size differential between native and de-sialylated protein is sufficient for detection. Warranties and liabilities Inc warrants that the above product conforms to the specifications described herein. Should the product fail for reasons other than through misuse Inc will, at its option, replace free of charge or refund the purchase price. This warranty is exclusive and Inc makes no other warrants, expressed or implied, including any implied conditions or warranties of merchantability or fitness for any particular purpose. Inc shall not be liable for any incidental, consequential or contingent damages. This product is intended for in vitro research only. revised on May 28, 2018 References Corfield, A. P., H. Higa, J. C. Paulson and R. Schauer. The specificity of viral and bacterial sialidases for alpha(2-3) and alpha(2-6)-linked sialic acids in glycoproteins. Biochim Biophys Acta 744: (1983). Dwek, R. A., C. J. Edge, D. J. Harvey, M. R. Wormald and R. B. Parekh. Analysis of glycoprotein-associated oligosaccharides. Ann Rev Biochem 62: (1993). Kobata, A. Use of endo- and exoglycosidases for structural studies of glycoconjugates. Anal Biochem 100:1-14 (1979). Ohta, Y., Y. Tsukada and T. Sugimori. Purification and properties of neuraminidase isoenzymes in Arthrobacter ureafaciens mutant. J Biochem (Tokyo) 106: (1989). Prime, S. J. Dearnley, A. M. Venton, R. B. Parekh and C. J. Edge. Oligosaccharide sequencing based on exo- and endoglycosidase digestion and liquid chromatographic analysis of the products. J Chromatogr A 720: (1996). Uchida, Y., Y. Tsukada and T. Sugimori. Enzymatic properties of neuraminidases from Arthrobacter ureafaciens. J Biochem (Tokyo) 86: (1979).

35 Sialadase Cp Alpha-(2-3,6) Nueraminidase, NANase Source recombinant from Clostridium perfringens Catalog Number E-S µl E-S µl E-S µl EC Specific Activity One unit of Sialidase is defined as the amount of enzyme required to produce 1 µmole of methylumbelliferone in 1 minute at 37 C, ph 5.0 from MU-NANA (2 -(4-methylumbelliferyl)-alpha-D-N acetylneuraminic acid]. Specificity All non-reducing terminal branched and unbranched a-(2-3) and a-(2-6) sialic acid. Relative activity a-(2-3) > a-(2-6) Applications Structural analysis of oligosaccharides Determining sialic acid linkage Glycoprotein deglycosylation Removing heterogeneity from glycoproteins Recommended Reagents included with E-S005: 1 vial: Reaction buffer 400 µl 250mM Sodium phosphate, ph 6.0 Activity 10 U/ml Specific Activity 250 U/mg Molecular Weight ~41,000 daltons ph optimum 6.0, active over the range mm sodium phosphate (ph 6.0) provides the optimal buffer for enzyme activity with sialyllactose, a standard substrate. If glycosidase treatment is performed at suboptimal ph because of glycoprotein solubility or activity requirements, expect some diminution in enzyme activity. Formulation The enzyme is provided as a sterile-filtered solution in 20 mm Tris-HCl, 25 mm NaCl (ph 7.5). Stability Stable at least 12 months when stored properly. Several days exposure to ambient temperatures will not reduce activity. Storage Store enzyme at 4 C. Do not freeze. Purity Sialidase Cp is tested for contaminating protease as follows: 10 µg of denatured BSA is incubated at 37 C for 24 hours with 2 µl of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation. The production host strain has been extensively tested and does not produce any detectable glycosidases. Sialidase Cp Specifications - Protocol

36 E-S005 Product Specifications - Protocol Directions for use 1. Add up to 100 µg of glycoprotein or 1 nmol of oligosaccharide to tube. 2. Add de-ionized water to a total of 14 µl. 3. Add 4 µl 5x Reaction Buffer Add 2 µl Sialidase Cp. 5. Incubate at 37 C for 1 hour. NOTE: longer incubation times are necessary if branched sialic acids are present. Desialylation may be monitored by SDS-PAGE if the size differential between native and de-sialylated protein is sufficient for detection. Warranties and liabilities, Onc warrants that the above product conforms to the specifications described herein. Should the product fail for reasons other than through misuse, Inc will, at its option, replace free of charge or refund the purchase price. This warranty is exclusive and, Inc makes no other warrants, expressed or implied, including any implied conditions or warranties of merchantability or fitness for any particular purpose., Inc shall not be liable for any incidental, consequential or contingent damages. This product is intended for in vitro research only. revised on May 28, 2018 References: Corfield, A. P., H. Higa, J. C. Paulson and R. Schauer. The specificity of viral and bacterial sialidases for alpha(2-3) and alpha(2-6)-linked sialic acids in glycoproteins. Biochim Biophys Acta 744: (1983). Dwek, R. A., C. J. Edge, D. J. Harvey, M. R. Wormald and R. B. Parekh. Analysis of glycoprotein-associated oligosaccharides. Ann Rev Biochem 62: (1993). Kobata, A. Use of endo- and exoglycosidases for structural studies of glycoconjugates. Anal Biochem 100:1-14 (1979). Prime, S. J. Dearnley, A. M. Venton, R. B. Parekh and C. J. Edge. Oligosaccharide sequencing based on exo- and endoglycosidase digestion and liquid chromatographic analysis of the products. J Chromatogr A 720: (1996). Roggentin, P, B. Rothe, F. Lottspeich and R. Schauer. Cloning and sequencing of a Clostridium perfringens sialidasegene. FEBS Lett 238: (Sept 1988). Roggentin P., R. G. Kleineidam and R. Schauer. Diversity in the properties of two sialidase isoenzymes produced by Clostridium perfringens spp. Biol Chem Hoppe-Seyler 376: (1995).

37 Sialadase Sp Alpha-(2-3) Nueraminidase, NANase Source recombinant from Streptococcus pneumoniae in E. Coli Catalog Number E-S µl E-S µl E-S µl EC Applications Structural analysis of oligosaccharides Determining sialic acid linkage Glycoprotein deglycosylation Removing heterogeneity from glycoproteins Recommended Reagents included with E-S007: 1 vial: Reaction buffer 400 µl 250mM Sodium phosphate, ph 6.0 Activity 10 U/ml Specific Activity 250 U/mg Molecular Weight ~75,000 daltons ph optimum 6.0, active over the range mm sodium phosphate (ph 6.0) provides the optimal buffer for enzyme activity with sialyllactose, a standard substrate. If glycosidase treatment is performed at suboptimal ph because of glycoprotein solubility or activity requirements, expect some diminution in enzyme activity. Specific Activity One unit of Sialidase is defined as the amount of enzyme required to produce 1 µmole of methylumbelliferone in 1 minute at 37 C, ph 5.0 from MU-NANA (2.-(4-methylumbelliferyl)-alpha-D-N acetylneuraminic acid]. Specificity All non-reducing terminal branched and unbranched a-(2-3) sialic acid. Formulation The enzyme is provided as a sterile-filtered solution in in 50 mm Sodium phosphate ph 7.5. Stability Stable at least 12 months when stored properly. Several days exposure to ambient temperatures will not reduce activity. Storage Store enzyme at 4 C. Do not freeze. Purity Sialidase Sp is tested for contaminating protease as follows: 10 µg of denatured BSA is incubated at 37 C for 24 hours with 2 µl of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation. The production host strain has been extensively tested and does not produce any detectable glycosidases. Sialidase Cp Specifications - Protocol

38 E-S007 Product Specifications - Protocol Directions for use 1. Add up to 100 µg of glycoprotein or 1 nmol of oligosaccharide to tube. 2. Add de-ionized water to a total of 14 µl. 3. Add 4 µl 5x Reaction Buffer Add 2 µl Sialidase Sp. 5. Incubate at 37 C for 1 hour. NOTE: longer incubation times are necessary if branched sialic acids are present. Desialylation may be monitored by SDS-PAGE if the size differential between native and de-sialylated protein is sufficient for detection. Warranties and liabilities, Onc warrants that the above product conforms to the specifications described herein. Should the product fail for reasons other than through misuse, Inc will, at its option, replace free of charge or refund the purchase price. This warranty is exclusive and, Inc makes no other warrants, expressed or implied, including any implied conditions or warranties of merchantability or fitness for any particular purpose., Inc shall not be liable for any incidental, consequential or contingent damages. This product is intended for in vitro research only. updated October 15, 2014 References: Corfield, A. P., H. Higa, J. C. Paulson and R. Schauer. The specificity of viral and bacterial sialidases for alpha(2-3) and alpha(2-6)-linked sialic acids in glycoproteins. Biochim Biophys Acta 744: (1983). Dwek, R. A., C. J. Edge, D. J. Harvey, M. R. Wormald and R. B. Parekh. Analysis of glycoprotein-associated oligosaccharides. Ann Rev Biochem 62: (1993). Kobata, A. Use of endo- and exoglycosidases for structural studies of glycoconjugates. Anal Biochem 100:1-14 (1979). Prime, S. J. Dearnley, A. M. Venton, R. B. Parekh and C. J. Edge. Oligosaccharide sequencing based on exo- and endoglycosidase digestion and liquid chromatographic analysis of the products. J Chromatogr A 720: (1996). Roggentin, P, B. Rothe, F. Lottspeich and R. Schauer. Cloning and sequencing of a Clostridium perfringens sialidasegene. FEBS Lett 238: (Sept 1988). Roggentin P., R. G. Kleineidam and R. Schauer. Diversity in the properties of two sialidase isoenzymes produced by Clostridium perfringens spp. Biol Chem Hoppe-Seyler 376: (1995) phone/fax info@.com phone fax

39 Endo-β-Galactosidase Source recombinant gene from Bacteroides fragilis in E.Coli EC Catalog Number E-XBG01 60 µl E-XBG µl E-XBG µl Recommended Reagents included with E-XBG01: 1 vial: Reaction buffer μl 250mM Sodium phosphate, ph 5.8 Activity 14 U/ml Specific Activity 140 U/mg ph Optimum 5.8 Molecular Weight 32,000 daltons Formulation The enzyme is provided as a sterile-filtered solution in 20 mm Tris-HCl, ph 7.5 Applications Endo-β-Galactoctosidase (EC ) cleaves internal β(1-4) galactose linkages in unbranched, repeating poly- N-acetyllactosamine structures. Sulfated structures such as keratan sulfate are also cleaved. Branching and/or fucosylation of the substrate may decrease or eliminate cleavage. Endo-β-Galactoctosidase is useful for identifying and removing poly-n-acetyllactosamine structures on many biologically important glycoconjugates. Specificity Internal β(1-4) galactose linkages in unbranched, repeating poly-n-acetyllactosamine [GlcNAcβ(1-3) Galβ(1-4)]n structures are the preferred substrate. Sulfated structures such as keratan sulfate are also cleaved. Branching and/or fucosylation of the substrate may decrease or eliminate cleavage. Sulfation of C-6 on galactose will block cleavage. Oligosaccharidesof the neo-lacto group are cleaved at greatly educed rates depending on the deviation from the preferred substrate. Storage Store enzyme at 4 C. Do not freeze. Stability Stable at least 12 months when stored properly. Several days exposure to ambient temperatures will not reduce activity. Active at least 5 days under reaction conditions. For example, Galβ(1-3)GlcNAcβ(1-3)Galβ(1-4)Glc is cleaved at 5x10-5 the rate of keratan sulfate(see ref.4). Specificity is similar to the Escherichia freundii enzyme. except that it is limited to cleaving N-acetyllactosamine extensions on tetraantennary structures of erythropoietin(see ref 5). Endo-β-Glycosidase Specifications - Protocol

40 E-G001 Product Specifications - Protocol Specific Activity One unit of endo-β-galactoctosidase is defined as the amount that will liberate one μmole of reducing sugar per minute at 37 o C and ph 5.8 from bovine corneal keratan sulfate. Purity Endo-β-Galactoctosidase is tested for contaminating protease as follows: 10 ug of denatured BSA is incubated for 24 hours at 37 o C with 2 μl of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation. The production strain of E. coli has been extensively tested and does not produce any detectable glycosidases. Directions for use For glycoproteins: 1. Add up to 100 μg of glycoprotein to a tube. 2. Add 4 ul 5X buffer and water to 19 μl. 3. Add 1 μl enzyme. 4. Incubate at 37 o C for 2 hrs. Procedure for oligosaccharides: Same as above except incubate from several hours to several days depending on the substrate. Add bovine serum albumen to 2 mg/ml to stabilize the protein during extended incubations. References 1. Scudder,P., Uemura, K., Doby, J., Fukuda, M.N. & Feizi, T.(1983) Isolation and characterization of an endo-β- galactosidase from Bacteroides fragilis Biochem. J. 213, Scudder,P., Hanfland, Pl, Uemura, K. & Feizi, T. (1984) Endo-β-galactosidases of Bacteroides fragilis and Escherichia freundii hydrolyze linear but not branched oligosaccharide domains of glycolipids of the neolacto series. J. Biol. Chem. 259, Scudder, P. Tang, P.W., Hounsell, E.F., Lawson, A.M., Mehmet, H. & Feizi, T. (1986) Isolation and characterization of sulfated oligosaccharides released from bovine corneal keratan sulphate by the action of endo-β-galactosidase. Eur. J. Biochem. 157, Murata, T., Hattori, T. Amarume, S. Koicki, A. & Usui, T. (2003) Kinetic studies on endo-βgalactosidase by a novel colorimetric assay and sythesis of N-acetyllactosaminerepeating oligosaccharide β-glycosides using its transglycosylation activity. Eur. J. Biochem 270, Hokke, C.H., Bergwerff, A.A., Van Dedem, D.W., Kamerling, J.P, and Vliegenthart, J.F. (1995) Structural analysis of the N- and O-linked carbohydrate chains of recombinant human erythropoietin expressed in Chinese hamster overay cells. Sialylation patters and branch location of dimeric N-acetyllactosamine units. Eur. J. Biochem. 228, Warranties and liabilities warrants that the above product conforms to the attached analytical documents. Should the product fail for reasons other than through misuse will, at its option, replace free of charge or refund the purchase price. This warranty is exclusive and makes no other warrants, expressed or implied, including any implied conditions or warranties of merchantability or fitness for any particular purpose. shall not be liable for any incidental, consequential or contingent damages. This product is intended for in vitro research only. revised on May 28, 2018

41 β-(1-2) Xylosidase Source Xanthomonas Catalog Number E-XYL01 Certification of Analysis Lot Number 511.1A Contents 1 vial: β-xylosidase- 60 µl (0.9 U) in 25 mm Tris-HCl, ph 7.5 and 25 mm NaCl 1 vial: Reaction buffer µl 250mM Sodium Acetate, ph 5 with 25 mm CaCl 2 Specific Activity >20 U/mg Activity 10 U/ml ph Optimum MW 100,000 daltons Formulation The enzyme is provided as a sterile-filtered solution in in 25 mm Tris-HCl, ph 7.5 and 25 mm NaCl Storage Store enzyme at 4 C. Do not freeze. Stability Stable at least 12 months when stored properly. Several days exposure to ambient temperatures will not reduce activity. Active at least 5 days under reaction conditions. Applications β (1 2) Galactoctosidase (EC ) cleaves xylose β(1-2) linked to the β-mannose of N-linked oligosacchardies. The α(1-3)-linked mannose must be first removed. Use α(1-2,3,6) Mannosidase (E-AM01) and α(1-6) core Mannosidase(E-AM02) to remove all mannose residues prior to Xylosidase treatment. Any other residues attached to the non-reducing edn of the α(1-3) mannose must also be removed. Specificity Non-reducing terminal beta-(1-2) xylose when linked to β- mannose when alpha-(1-3) mannose has been removed. Specific Activity One unit of Beta-(1-2)-Xylosidase is defined as the amount of enzyme required to produce 1 nmole of methylumbelliferone in 1 minute at 37 C ph 5 with 5 mm calcium chloride from 4- methlyumbellifery-7-beata-d-xyloside. Purity β-xylosidase is tested for contaminating protease as follows: 10 ug of denatured BSA is incubated for 24 hours at 37 o C with 2 µl of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation. E-XBG01 β-(1-2) Xylosidase Specifications - Protocol Eaton Road San Mateo, CA info@.com fax

42 E-XYL 01 Product Specifications - Protocol Each lot is tested for contaminating activities by incubating the enzyme for 24 hours with the appropriate substrates: the detection limit of these assays is 5 uu/ml (IUB). A passing lot will have no detectable activity. Directions for use For glycoproteins: 1.Add 1 µl of enzyme to 1 nmol of oligosaccharide. 2. Add 4 ul 5X buffer and water to 19 µl. 4. Incubate at 37 o C for 18 hrs. For oligosaccharides: Same as above except incubate from several hours to several days depending on the substrate. Warranties and liabilities warrants that the above product conforms to the specifications described herein. Should the product fail for reasons other than through misuse will, at its option, replace free of charge or refund the purchase price. This warranty is exclusive and makes no other warrants, expressed or implied, including any implied conditions or warranties of merchantability or fitness for any particular purpose. For research use only Updated December 9, Eaton Road San Mateo, CA info@.com fax

43 Enzymatic CarboRelease Kit Part Number KE-DG01 Kit Storage Kits should be stored at 4 C. Shipping This product should be shipped on frozen packs in an insulated container. Kit Contents Kit includes the enzymes, controls, and reagents required to remove all N-linked oligosaccharides and most O-linked sugars. Each kit will deglycosylate more than 2 mg of glycoprotein in 20 reactions. Enzymes 20 µls of each of the following enzymes in separate vials: PNGase F (E. meningosepticum) O-Glycosidase (S. pneumoniae) Sialidase (A. ureafaciens) ß-Galactosidase (S. pneumoniae) Glucosaminidase (S. pneumonia) refer to enzyme specifications for further details Control Fetuin is include in this kit as a positive control of the deglycosylation reaction. The concentration of the fetuin is 10 mg/ml.the molecular weight is approximately 48,000 daltons. Specificity The Enzymatic CarboRelease Kit will remove all N-linked oligosaccharides and many O-linked oligosaccharides from glycoproteins. N-links (Asn-linked) are removed using the enzyme PNGase F. In addition, all Ser/Thrlinked (O-linked) Gal-(β1-3)-GalNAc-(α1) and all sialic acid substituted Gal-(β1-3)-GalNAc-(α1) will be removed using the combination of Sialidase and O-Glycosidase. The addition of ß-Galactosidase and Glucosaminidase will assist in the deglycosylation of larger O-link structures. Other Supplied Reagents 5x Reaction buffer µl 250 mm sodium phosphate, ph 7 Denaturation Solution µl Triton X µl Bovine Fetuin (control) - 10 mg/ml KE-DG01 CarboRelease Kit Specifications - Protocol

44 KE-DG01 Product Specifications - Protocol Directions for Use 1. Mix 10 µl of 5x reaction buffer with up to 100 µg of glycoprotein in 30 µl distilled water in a 1.5 ml tube. 2. Add 2.5 µl denaturation solution. Mix gently and place in boiling water bath for 5 minutes. Chill on ice. 3. Add 2.5 µl of Triton-X. 4. Add 1 µl each of PNGase F, Sialidase, ß-Galactosidase, Glucosaminidase, and O-Glycosidase. Incubate for 3 hours at 37 C. Note: Denaturation increases the rate of enzyme digestion up to 10 fold. If denaturation is not desired omit step 2-3, add with 5 µl of distilled water and increase incubation time to 24 hours. The efficiency of deglycosylation can be tested by running a sample on a SDS-PAGE gel. Warranties and liabilities warrants that the above product conforms to the attached analytical documents. Should the product fail for reasons other than through misuse will, at its option, replace free of charge or refund the purchase price. This warranty is exclusive and makes no other warrants, expressed or implied, including any implied conditions or warranties of merchantability or fitness for any particular purpose. shall not be liable for any incidental, consequential or contingent damages. This product is intended for in vitro research only. updated 11/24/ phone/fax info@.com phone fax

45 Enzymatic DeglycoMx Kit Part Number KE-DGMX Kit Storage Kits should be stored at 4 C. Kit Contents Kit includes the enzymes, and reagents required to remove all N-linked oligosaccharides and most O-linked sugars. Each kit will deglycosylate more than 0.5 mg of glycoprotein in 10 reactions. Enzymes The enzymes are provided as one 20 µl premixed cocktail including: PNGase F (E. meningosepticum) 25 mu O-Glycosidase (S. pneumoniae) 6.25 mu Sialidase (A. ureafaciens) 25 mu ß-Galactosidase (S. pneumoniae) 15 mu Glucosaminidase (S. pneumonia) 5 mu refer to enzyme specifications for further details Other Supplied Reagents 5x Reaction buffer µl 250 mm sodium phosphate, ph 7 Denaturation Solution µl Triton X µl Specificity The Enzymatic DeGlycoMx Kit will remove all N-linked oligosaccharides and many O-linked oligosaccharides from glycoproteins. N-links (Asnlinked) are removed using the enzyme PNGase F. In addition, all Ser/Thr-linked (O-linked) Gal-(β1-3)- GalNAc-(α1) and all sialic acid substituted Gal-(β1-3)- GalNAc-(α1) will be removed using the combination of Sialidase and O-Glycosidase. The addition of ß-Galactosidase and Glucosaminidase will assist in the deglycosylation of larger O-link structures. Directions for Use 1. Mix 10 µl of 5x reaction buffer with up to 50 µg of glycoprotein in 33 µl distilled water in a 1.5 ml tube. 2. Add 2.5 µl denaturation solution. Mix gently and place in boiling water bath for 5 minutes. Chill on ice. 3. Add 2.5 µl of Triton-X. 4. Add 2 µls of the DeGlycoMx enzyme cocktail. Incubate for 3 hours at 37 C. Note: Denaturation increases the rate of enzyme digestion up to 10 fold. If denaturation is not desired omit step 2-3, add with 5 µl of distilled water and increase incubation time up to 24 hours. The efficiency of deglycosylation can be tested by running a sample on a SDS-PAGE gel. Warranties and liabilities warrants that the above product conforms to the attached analytical documents. Should the product fail for reasons other than through misuse will, at its option, replace free of charge or refund the purchase price. This warranty is exclusive and QA- Bio makes no other warrants, expressed or implied, including any implied conditions or warranties of merchantability or fitness for any particular purpose. shall not be liable for any incidental, consequential or contingent damages. This product is intended for in vitro research only. revised on May 29, 2018 KE-DGMX DeGlycoMx Kit Specifications - Protocol

46 Endo F Multi-Kit includes: Endo F1, Endo F2, Endo F3 Source recombinant Elizabethkingia meningosepticum in E. Coli (was Chryseobacterium meningosepticum) Catalog Number KE-EFX3 EC Contents 1 vial: Endo F1-20 μl (0.3 U) 20 mm Tris-HCl ph vial: Endo F2-20 μl (0.1 U) 10 mm sodium acetate, 25 mm NaCl, ph vial: Endo F3-20 μl (0.1 U) 20 mm Tris-HCl ph vial: 5x Reaction Buffer μl 250 mm sodium acetate, ph4.5 1 vial: 5x Reaction Buffer μl 250 mm sodium phosphate, ph5.5 Specific activity Endo F1 16 U/mg Endo F2 20 U/mg Endo F3 25 U/mg Activity Endo F1 17 U/ml Endo F2 5 U/ml Endo F3 5 U/ml Specific Activity Defined as the amount of enzyme required to catalyze the release of N-linked oligosaccharides from 1 micromole of denatured Ribonuclease B (Endo F1) or porcine fibrinogen peptides (Endo F2/F3) in 1 minute at 37 C, ph 5.5 (PH 4.5 for Endo F3). Cleavage is monitored by SDS-PAGE. Formulation The enzymes are provided as a sterile-filtered solution. Storage Store enzyme at 4 C. Do not freeze. Stability Several days exposure to ambient temperatures will not reduce activity. Stable at least 12 months when stored properly. KE-EFX3 Endo F Multi-Kit Specifications - Protocol