Releases α(2,3)-, α(2,6)-, α(2,8)-, and α(2,9)-linked N-acetylneuraminic acid from oligosaccharides and glycoproteins. Also capable of releasing N-glycolylneuraminic acid.
Recombinant gene from Arthrobacter ureafaciens, expressed in E. coli.
The enzyme releases α(2,3)-, α(2,6)-, α(2,8)-, and α(2,9)-linked N-acetylneuraminic acid from complex carbohydrates. The initial rate of hydrolysis of α(2,6) linkages is reported to be approximately twice that of α(2,3)-linked sialic acid however, in practice, this kinetic selectivity is of little consequence during extended incubations. Effective digestion of glycolipid substrates is facilitated by addition of a detergent, such as sodium taurodeoxycholate to the incubation.
Sialidase A is capable of releasing N-glycolylneuraminic acid (Neu5Gc, NGNA) in addition to N-acetylneuraminic acid (Neu5Ac, NANA) , although similarly to other sialidases  the activity is lower toward Neu5Gc than Neu5Ac.
The enzyme can also be used to remove sialic acid from gangliosides , glycosphingolipds (ceramide & oligosaccharide) with sialic acid.
May be used for exoglycosidase sequencing in conjunction with Sialidase S (GK80021), which is specific for α(2,3)-linked sialic acids.
Sialidases are also known as neuraminidases.
WS0049 5x Reaction Buffer B [250 mM sodium phosphate pH 6.0]
5X concentrated buffer which when diluted gives 50 mM sodium phosphate pH 6.0.
20 mM Tris HCl pH 7.5, containing 25 mM NaCl.
One unit is defined as the amount of enzyme required to catalyze the release of 1 μmole of p-nitrophenol from p-nitrophenyl-α-D-N-acetylneuraminic acid per minute at 37° C, pH 5.5.
Size: 1 U (200 µl)
Concentration: ≥ 5 U/ml
Product Code: GK80040
Q. Does GK80040 contain a purification tag, such as 6xHis?
A. No. Although GK80040 Sialidase A is expressed recombinantly, it does not contain a purification tag.
Q. I do not want to use the sodium phosphate reaction buffer supplied with GK80040. Can I use an acetate buffer? Is this good for digestion with multiple exoglycosidases?
A. We use an ammonium acetate buffer when we run multi-enzyme exoglycosidase digests that include GK80040 Sialidase A. We suggest a 10X reaction buffer of 500 mM ammonium acetate pH 5.5 w/ 0.05% azide. The reaction buffer can also be used diluted to 20X (25 mM ammonium acetate) and lower with purified glycans. This buffer works with most of our exoglycosidases in an overnight digestion:
|Product Code||Product Name||Source||Specificity|
|GK80040||Sialidase A||Recombinant gene from Arthrobacter ureafaciens, expressed in E.coli||Releases non-reducing terminal α(2-3,6,8,9)-linked sialic acid|
|GK80021||Sialidase S||Recombinant from Streptococcus pneumoniae, expressed in E. coli (6 x His tagged)||Releases non-reducing terminal α(2-3)-linked sialic acid|
|Releases non-reducing terminal β(1-4)-linked galactose|
|GKX-5013||β(1-3,4) Galactosidase||Bovine testis||Releases non-reducing terminal β(1-3,4)-linked galactose|
|Green coffee bean||Releases non-reducing terminal α(1-3,4,6)-linked galactose (alpha gal)|
|GKX-5003||β(1-2,3,4,6) Hexosaminidase||Jack bean||Releases non-reducing terminal β(1-2,3,4,6)-linked N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) residues|
|Jack bean||Releases non-reducing terminal β(1-2,3,4,6)-linked GlcNAc and GalNAc residues, higher concentration than GKX-5003 for improved release of bisecting GlcNAc|
|Recombinant gene from Streptococcus pneumoniae, expressed in E. coli||Cleaves all non-reducing terminal β-linked GlcNAc, but not bisecting β(1-4) GlcNAc. Does not release GalNAc|
|Jack bean||Releases non-reducing terminal α(1-2,3,6)-linked mannose, with kinetic preference for α(1-2,3)-linked mannose over α(1-6)-linked mannose.|
|Aspergillus saitoi||Releases non-reducing terminal α(1-2)-linked mannose, high mannose N-glycan structures are digested to Man5|
Generally, 2 μl of each exoglycosidase is used in a 20-30 μl reaction with an overnight incubation (16 hours) at 37°C. A general example is shown below, please Contact Us for more details.
|Reagent (µl)||Rxn 1||Rxn 2||Rxn 3||Rxn 4||Rxn 5|
|Labeled glycans in buffer||20||20||20||20||20|
|Sialidase A* [GK80040]||-||2||2||2||2|
|β-Galactosidase [GKX-5013 or GKX-5014]||-||-||2||2||2|
|β N-acetylhexoseaminidase [GKX-5023]||-||-||-||2||2|
*If sialic acid linkage determination is needed, Sialidase S [GK80021] cleaves α(2-3)-linked sialic acid may also be used in a separate reaction from sialidase A, which cleaves α(2-3,6,8,9)-linked sialic acids.
For data using these methods see our poster, Characterizing Low-Abundance Glycans on Therapeutic Proteins, and our WCBP 2016 presentation.
GKE-5006 α(1-2,3,4,6) fucosidase (bovine kidney) is unlikely to release core α1,6) fucose from the innermost GlcNAc of N-glycans if there is a fluorophore attached to GlcNAc. If possible, fucosidase digestion to remove core fucose should be performed on released N-glycans prior to labeling.
Acetate buffers work better than phosphate for injecting onto LC (phosphate phase separates in acetonitrile), and acetate is volatile so samples can be dried down if necessary.
Q. Can GK80040 be used with detergents and reductants?
A. The enzyme can be used with sodium dodecyl sulfate (SDS) and β-mercaptoethanol (βME) at certain levels.
The 5x Denaturation Solution used in the GK80110 kit is 2% sodium dodecylsulfate (SDS) and 1 M βME, and 2.5μl is used in a reaction volume of around 50μl giving a final reaction concentration of 0.1% SDS, 50mM bME. Denaturation Solution and 5x Incubation Buffer are added to the glycoprotein substrate and the mixture is heated to 100°C for 5 minutes. After cooling, 2.5μl of a Detergent Solution (15% NP-40, final reaction concentration 0.75%) is added to counteract the inhibitory effect of SDS on PNGase F. This is followed by the addition of N-Glycanase, Sialidase A (GK80040) and O-Glycanase (GK80090), with incubation for 3 hours at 37°C.
Product Safety Documentation for GK80040:
|GK80040||Glyko® Sialidase A™|
|WS0049||5x Reaction Buffer B|