Detailed Characterization of Several Glycosidase Enzymes
β1-3 Galactosidase
The GlcNac (β1-6) residue is the only anomeric configuration that can effect the specificity of the enzyme enabling cleavage of the non-reducing β1-4 Galactose.
Selling concentration of the enzyme will cut the β1-4 Galactose linkage as shown in (A) due to the adjacent GlcNAcβ1-6 anomer. This cleavage will not occur if the selling concentration of the enzyme is diluted 16-fold, shown in (B).
Chart Legend: Gal Glc
Man
GalNAc
GlcNAc
Fuc
NeuAc
R = any sugar
![](/en-us/-/media/nebus/page-images/tools-and-resources/usage-guidelines/fig1a.jpg?rev=e6d43a8d49094bdbaa009fe35ecd600d&hash=B6889873C26F932EDEAC497F89A98307)
![](/en-us/-/media/nebus/page-images/tools-and-resources/usage-guidelines/fig1b.jpg?rev=2c19ab714ee9437199ca05cc68b54f0a&hash=95250523765F2D7713A5F7DC8B0232C0)
β1-4 Galactosidase
Reactions (A), (B) and (C) contained 2 units, 4 units and 8 units of β1-4 Galactosidase, respectively, and either 1X G4 or 1X G6 reaction buffer in a total reaction volume of 10 µl. Reactions were incubated at 37°C.
![](/en-us/-/media/nebus/page-images/tools-and-resources/usage-guidelines/fig2a.jpg?rev=5d54bece7c7142449ab2e960640972c6&hash=BF89ED15D3D421A8E321EF82B48A799D)
![](/en-us/-/media/nebus/page-images/tools-and-resources/usage-guidelines/b14galactosidase_b.jpg?rev=760488def4924ad48c2fb5361d0cd411&hash=3A217ABBBF459923841B78FADB086AE9)
![](/en-us/-/media/nebus/page-images/tools-and-resources/usage-guidelines/fig2c.jpg?rev=791b1253bdb347a2aeda18a9b430500d&hash=C8B99E03D5C006169F1E6EE36794FA73)
β-N-Acetylglucosaminidase
All reactions contained 4 units of β-N-Acetylglucosaminidase, 1X G1 reaction buffer and 1X BSA in a total reaction volume of 10 µl. Reactions (B), (C) and (D) were treated with 8 units of β1-4 Galactosidase prior to treatment with β-N-Acetylglucosaminidase. Reactions were incubated at 37°C.
![](/en-us/-/media/nebus/page-images/tools-and-resources/usage-guidelines/fig3a.jpg?rev=2d84c28c02a94766bfef0433a5f0fd43&hash=2D70BF731A4279D50C3302F68F18CA7D)
![](/en-us/-/media/nebus/page-images/tools-and-resources/usage-guidelines/fig3b.jpg?rev=b95771481afd499b9c4ea05fa8b87552&hash=B46067DCC52776F1D4EBF683A9C83C8E)
![](/en-us/-/media/nebus/page-images/tools-and-resources/usage-guidelines/bn_acetylglucosaminidase_c.jpg?rev=5ea1c82c84dc4b0fa3b416c5c75096a1&hash=67AFEECAF347A4020DFE8E25BDA73EEC)
![](/en-us/-/media/nebus/page-images/tools-and-resources/usage-guidelines/bn_acetylglucosaminidase_d.jpg?rev=dbaa0215057844ff91679c2958eb2017&hash=BC08249B4A0A7FEB5C7AB1D5A45AB367)
α1-2,3 Mannosidase
All reactions contained 32 units of α1-2,3 Mannosidase, 1X G6 reaction buffer and 1X BSA in a total reaction volume of 10 µl. Reactions were incubated at 37°C.
![](/en-us/-/media/nebus/page-images/tools-and-resources/usage-guidelines/fig4a.jpg?rev=c3776649f4b74a9a93232b7da6b4fd76&hash=1074405284F8D4C04EF7F0EF80EAD9C5)
![](/en-us/-/media/nebus/page-images/tools-and-resources/usage-guidelines/fig4b.jpg?rev=b16ff09aa65e479eb7f61c82bd907967&hash=DFD2193F5C2A7426FED59B5599EFCFE6)
![](/en-us/-/media/nebus/page-images/tools-and-resources/usage-guidelines/fig4c.jpg?rev=8671a27a7040465c991b1ca6997a73e9&hash=3AEE2EA3FA883803E106AC9F08C37D7F)
![](/en-us/-/media/nebus/page-images/tools-and-resources/usage-guidelines/fig4d.jpg?rev=2aef7987326a456fa0244615daf61a27&hash=38D52D759A6C5004564F81EB20BD6422)
α1-6 Mannosidase
All reactions contained 32 units of α1-2,3 Mannosidase (NEB #P0729), 40 units of α1-6 Mannosidase, 1X G6 reaction buffer and 1X BSA in a total reaction volume of 10 µl. Reactions were incubated at 37°C. The substrate depicted in (E) will not cut to completion. If this structure exists in any substrate it will be impervious to cleavage by α1-6 Mannosidase. Note: When used alone, α1-6 Mannosidase will still act only on linear substrates. When used in conjunction with α1-2,3 Mannosidase, the α1-6 Mannosidase will cleave α1-6 Mannose residues from branched carbohydrate substrates.
![](/en-us/-/media/nebus/page-images/tools-and-resources/usage-guidelines/fig5a.jpg?rev=a52e103d109c4ef9bd36f3849da96a23&hash=FF1C4640E47E1E895F1AF70BA7118568)
![](/en-us/-/media/nebus/page-images/tools-and-resources/usage-guidelines/fig5b.jpg?rev=d6ffeaa0581d45a6bea6b39ad5b8896f&hash=C02CBB5B6F9812C82D71D0E51CB92F95)
![](/en-us/-/media/nebus/page-images/tools-and-resources/usage-guidelines/fig5c.jpg?rev=92db7d0e0a3e47fa93b29f01fc45e18c&hash=47F3DE90EAFD5AC0989BE855E11185E7)
![](/en-us/-/media/nebus/page-images/tools-and-resources/usage-guidelines/fig5d.jpg?rev=78848ad30ad74eea9ec948c3a9730531&hash=2105434C3C53625E61193E6D95F200FF)
![](/en-us/-/media/nebus/page-images/tools-and-resources/usage-guidelines/fig5e.jpg?rev=39b7f2f947064976a385593959ab3c3d&hash=64B3132DF3A1B8A1F7C027889BAC60A5)
α1-3,6 Galactosidase
Reaction (A) contained 24 units of α1-3,6 Galatosidase and 100 units of Neuraminidase (NEB #P0720), followed by a heat kill at 65°C for 10 minutes and a 2 hour digestion with 16 units of β1-4 Galactosidase (NEB #P0730).
The reaction in (B) contained 4 units of α1-3, 6 Galactosidase, 1X G6 reaction buffer and 1X BSA in a total reaction volume of 20 µl.
![](/en-us/-/media/nebus/page-images/tools-and-resources/usage-guidelines/a1_3_6_galactosidase_a.jpg?rev=53ef5d9d8e4a476dbd91f0f571fb9992&hash=956E58576D092988A8E96D18A3217FA5)
![](/en-us/-/media/nebus/page-images/tools-and-resources/usage-guidelines/a1_3_6_galactosidase_b.jpg?rev=e2b4a720317c41e8884b0995b55d765a&hash=8602A59EAE5D1C3DAFDFB441684CBBCC)
α1-2 Fucosidase
Reactions (A), (B) and (D) contained 1X G4 reaction buffer and reaction (C) contained 1X G6 reaction buffer. All reactions contain 1X BSA in a total reaction volume of 10 µl and all reactions were incubated at 37°C. All reactions contained 20 units of α1-2 Fucosidase. Reaction (C) also contained 20 units of α-N-Acetylgalactosaminidase (NEB #P0734). In reaction (C), the branched α1-2 fucose is removed in the presence of both enzymes, but not by α1-2 Fucosidase alone.
![](/en-us/-/media/nebus/page-images/tools-and-resources/usage-guidelines/fig7a.jpg?rev=2003aa1fa5e34709a5ae2fda8f7c4552&hash=07614A65E0D1E16FDF306CCF75B982E5)
![](/en-us/-/media/nebus/page-images/tools-and-resources/usage-guidelines/fig7b.jpg?rev=ae60b77319eb4332a2f0434c5c66f86c&hash=CB8F4E9043AE1AAEB72E0DEF058B7B3E)
![](/en-us/-/media/nebus/page-images/tools-and-resources/usage-guidelines/a1_2_fucosidase_c.jpg?rev=d38426cdddd549d6ace95a45c081cd34&hash=BA005C1C3C9BF34615D839BE6363E6EE)
![](/en-us/-/media/nebus/page-images/tools-and-resources/usage-guidelines/fig7d.jpg?rev=562f7b737995443188c78d35a4c5d5d6&hash=591344EBC3B27D10E70C5DAF7BE10319)
PNGase F
PNGase F is not able to cleave N-linked glycans from glycoproteins when the innermost GlcNAc residue is linked to an α1-3 Fucose residue (1). This modification is most commonly found in plant and some insect glycoproteins.
(1) Tretter, V. et al. (1991) Eur. J. Biochem. 199, 647–652. PMID: 1868849
![](/en-us/-/media/nebus/page-images/tools-and-resources/usage-guidelines/fig8a.jpg?rev=93200c8d5b7543a69e3fb1c97f567272&hash=8C0B5E55870F4CE5EA710C7E7DE1717A)
![](/en-us/-/media/nebus/page-images/tools-and-resources/usage-guidelines/fig8b.jpg?rev=10f0eb549416414e8f2fde8cccb137f5&hash=5975B27FECED2FE0389D7A0CA32E50C9)
Remove-iT™ Endo S
Remove-iT Endo S does not have a strict peptide requirement for activity, thus the “X” can be a protein, peptide, Asparagine, or free glycan. Remove-iT Endo S is active on a substrate with or without core a(1-6)fucosylation as well as with or without a bisecting N-acetylglucosamine. Triantennary and tetrantennary sialyted or asialo glycans are not a substrate for Remove-iT Endo S.
![](/en-us/-/media/nebus/page-images/tools-and-resources/usage-guidelines/remove_it_endos.jpg?rev=8d8f5122c02340a7a5776dd2cca6b934&hash=18193892E1A9596126095F44CE9BBF53)
Remove-iT™ Endo D
Remove-iT Endo D, also known as Endoglycosidase D, is a recombinant glycosidase, which cleaves within the chitobiose core of paucimannose N-linked glycans, with or without extensions in the antennae. Remove-iT Endo D is tagged with a chitin binding domain (CBD) for easy removal from a reaction, and is supplied glycerol-free for optimal performance in HPLC- and MS- intensive methods.
![](/en-us/-/media/nebus/page-images/tools-and-resources/usage-guidelines/remove_it_endod.jpg?rev=ed8c155236d04140973b1ac6354306ac&hash=D1296040FC53E8243F754D4FF7DAF3E3)
O-Glycosidase (Endo-a-N-Acetylgalactosaminidase)
Reactions (A) and (B) contained 40,000 units of Endo-a-N-Acetylgalactosaminidase, 100 units of Neuraminidase and 1X G7 reaction buffer in a total reaction volume of 100 μl. The Reactions were incubated at 37°C and released O-linked disaccharide carbohydrates were determined by Morgan and Elson Assay.
![](/en-us/-/media/nebus/page-images/tools-and-resources/usage-guidelines/o_glycosidase_a.jpg?rev=2b7b7d9a081b47b2ace0fdd15bf0cc54&hash=D057FDC0886AB07CB512CF76A5F89062)
![](/en-us/-/media/nebus/page-images/tools-and-resources/usage-guidelines/o_glycosidase_b.jpg?rev=1ed7b055ffbd48e29ccbd2688a80e1f2&hash=A8E3A6C587C14B14EA0B73551A5188B1)