N-glycan Labeling and Detection Kit

The N-glycan Labeling and Detection Kit Insert (Catalog # EA007) is designed for the quick labeling and detection of N-glycans on various glycoproteins as well as studying the level of sialyation on glycoproteins. The kit provides all the necessary reagents for 100 labeling assays using the provided assay protocol. After optimization, the kit could allow for far more assays. This kit supplies Cy3-Neu5Ac Labeled ACE-2 Protein (Cy3-ACE2), with its N-glycans modified with Cy3-Neu5Ac. In addition, unlabeled Recombinant Human ACE-2 Protein (ACE2) is included as a model protein for the labeling activities of the kit. Used together they provide excellent controls for both the labeling efficiency of the kit and as a gel loading control.

N-glycan Labeling and Detection Kit

Features

• Enzymatic labeling under complete physiological conditions
• Labeling is highly specific to N-glycans
• Labeled proteins can be monitored via gel electrophoresis
• Labeled glycans can be easily released from samples by PNGase F treatment (See EA006)
• Labels can be replaced with other types of fluorophores, such as Cy5, by switching CMP-Cy3-Sialic Acid to CMP-Cy5-Syalic Acid (ES302) or biotin with CMP-C9-Biotin-Sialic Acid (ES201)
• Convenient procedure with hands on time less than 10 minutes

Preparation and Storage

Stability & Storage
Store the unopened product at -70 °C. Use a manual defrost freezer and avoid repeated freeze-thaw cycles. Do not use past expiration date.

Background: N-glycan Labeling and Detection Kit 

Glycosylation is commonly found on membrane and secreted proteins of eukaryotic cells. N- and O-glycans are among the most common glycans. N-glycans refer to glycans attached to the asparagine residues in the sequon of NxS/T (Asn-X-Ser/Thr, where X can be any amino acid except Pro) of a glycoprotein. N-glycans are initiated as high-mannose glycan in endoplasmic reticulum and are converted to hybrid and complex N-glycans during the maturation process in the Golgi apparatus. In hybrid N-glycan, terminal mannose residues in one branch of the glycan are replaced with a GlcNAc residue. In complex N-glycans, all terminal mannose residues are replaced with GlcNAc residues. The GlcNAc residues are usually elongated with Gal residues that are further terminated with sialic acids. O-glycans usually refer to O-GalNAc and all its derivatives. O-glycans are initiated by attaching a single GlaNAc residue to a Ser/Thr residues on proteins via polypeptide GalNAc transferases (ppGalNAcTs). O-GalNAc can be further extended by other sugar residues and become different subtypes, such as Core-1 and Core-2 O-glycans. In some glycoproteins, glycan components can account to more than 50% of their mass. Despite the abundance, labeling and detection of these glycans are extremely difficult.
 
This kit offers reagents for N-glycan labeling. N-glycan specific labeling is achieved through direct incorporation of Cy3-conjugated Neu5Ac (Cy3-Neu5Ac) or replacement of existing sialic acids with Cy3-Neu5Ac via N-glycan specific sialyltransferase ST6Gal1 (Fig. 1). Following the provided Assay Protocol, the kit provides sufficient reagents to perform 100 N-glycan labeling reactions. In addition, recombinant human ACE-2 is provided as assay control and Cy3-Neu5Ac labeled recombinant human ACE-2 (Cy3-ACE-2) is provided as a gel control.
 
In addition to N-glycan/glycoprotein labeling and detection, the kit can also be used to evaluate sialylation levels on glycoproteins. 
 
N-glycans can be labeled with other types of conjugated sialic acids, such as CMP-Cy5-Sialic Acid (Catalog # ES302) and CMP-C9-Sialic Acid (Catalog # ES201). Please check our website for the availability of any new activated conjugated sialic acids.