N-glycan Labeling and Detection Kit Summary
Learn more about Fluorescent Glycan Labeling and Detection
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
- 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.
- ST6Gal1 Protein
- Neuraminidase Protein
- Cy3-Neu5Ac Labeled ACE-2 Protein
- ACE-2 Protein
- 6X SDS Gel Loading Dye
- Glycan Labeling Assay Buffer (10X)
Strategy for N-glycan labeling. Glycan labeling is achieved through direct incorporation of Cy3-conjugated Neu5Ac (Cy3-Neu5Ac) (A) or replacement of natural sialic acids with Cy3-Neu5Ac (B). In both strategies, Cy3-Neu5Ac is introduced via recombinant ST6Gal1. In strategy B, samples are first desialylated with recombinant C. perfringens Neuraminidase (Neu) that specifically removes alpha 2-3 and alpha 2-6 linked sialic acids. Alternative fluorophore conjugated Neu5Ac donor substrates and sialyltransferases may be used for the labeling.
Specific detection of N- and O-glycans on different glycoproteins. Recombinant Human CEACAM-7 Protein (9010-CM), Recombinant Human CEACAM-8/CD66b Protein (9639-CM), Recombinant Human MUC-1 Fc Chimera Protein (10332-MU), and Recombinant Human CA125/MUC16 Protein (5609-MU) were labeled on N-glycan (N) by ST6Gal1 with Cy3-Neu5Ac according to the protocol and on O-Glycan (O) by Recombinant Human ST3GAL2 Protein (7275-GT) with Cy5-Neu5Ac and then separated on 4-20% gradient SDS-PAGE gel. Only N-glycans were detected on CEACAM-7 and CEACAM-8, and only O-glycans were detected on MUC-1, both N- and O-glycans were detected on MUC16. The left part is the TCE image of the gel and the right part is the fluorescent image.
N-glycan and O-glycan specific labeling of recombinant human ACE-2. N-glycan labeled recombinant human (rh) ACE-2 was prepared according to the provided protocol and O-glycan labeled rhACE-2 was prepared in the same way except that ST6Gal1 was replaced with O-glycan specific ST3Gal2. The labeled samples were then treated with 10-fold serial dilution of PNGase F starting with 100 ng using the PNGase F N-glycan Releasing Kit (EA006). The digestions were separated on 17% SDS-PAGE gel. The upper part is the fluorescent image and the lower part is the TCE image of the gel. While all labeled N-glycans could be released by PNGase F N-glycan Releasing Kit, no labeled O-glycans were released by PNGase F, demonstrating the specific labeling on N- and O-glycans by ST6Gal1 and ST3Gal2, respectively.
N-glycan and O-glycan specific labeling of Recombinant Human MUC16. Recombinant Human CA125/MUC16 Protein (5609-MU) was prepared according to the above protocol except that Cy3 was replaced with Cy5 in CA125/MUC16 and ST6Gal1 was replaced with ST3Gal2 for O-glycan labeling. The labeled samples were treated with 4-fold serial dilution of PNGase F starting with 640 ng using the PNGase F N-glycan Releasing Kit (EA006). The digestions were separated on 17% SDS-PAGE gel. The upper part is the fluorescent image and the lower part is the TCE image of the gel. While all labeled N-glycans could released by PNGase F N-glycan Releasing Kit, no labeled O-glycans were released by PNGase F, demonstrating the specific labeling on N- and O-glycans by and ST6Gal1 and ST3Gal2, respectively.
Determining the optimal pH for ST6Gal1 labeling. Optimal pH determination of a sialyltransferase was an extremely difficult task in the past. Using the N-glycan Labeling and Detection Kit (Catalog # EA007), this task becomes rather easy. In this figure, Recombinant Human ACE-2 was labeled by ST6Gal1 at different pH following the provided protocol except with different buffers. The labeled reactions were then separated on a 4-20% gradient SDS-PAGE. Both TCE protein image and fluorescent image are shown. Incorporation of Cy3 to rhACE-2 resulted in intensified band absorption in TCE images. While ST6Gal1 is active from pH 4.5-10, its optimal pH ranges from 5-9.
Effect of neuraminidase treatment on N-glycan labeling. Recombinant Human ACE-2 Protein (933-ZN) and Recombinant Human CA125/MUC16 Protein (5609-MU), without or with Neuraminidase (Neu) treatment, were labeled by ST6Gal1, then digested with PNGase F N-glycan Releasing Kit (EA006) and finally separated on 17% SDS-PAGE gel. Both the protein image stained with TCE (upper panel) and the fluorescent image (lower panel) are shown. The freed glycans are visible in both images. While Neuraminidase treatment slightly increased labeling on ACE-2, it greatly enhanced the labeling on MUC16, indicating that the majority N-glycans on MUC16 were initially sialylated. This data demonstrates that the kit is also a good tool to evaluate the sialylation levels on target proteins.
Effect of neuraminidase treatment on N-glycan labeling. A collection of glycoproteins were labeled on O-glycans with Cy3-Neu5Ac (green) by ST3Gal2 and N-glycans with Cy5-Neu5Ac (red) by ST6Gal1. The left side are gel images of labeled samples without PNGase F treatment. The right side are gel images of the samples after treatment with PNGase F N-glycan Releasing Kit (EA006). While MUC-1 mainly contains O-glycans and CD19 mainly contains N-glycans, the other proteins contain both N- and O-glycans at different levels. All labeled N-glycans (in red) except that on MUC-1 could be released by PNGase F N-glycan Releasing Kit, further confirming the specific labeling. The nature of the labeling on MUC-1 by ST6Gal1 is not clear. All samples were separated on 17% SDS-PAGE gel. Recombinant SARS-CoV-2 Spike RBD His-tag Protein (10500-CV), Recombinant Human Erythropoietin/EPO Protein, Recombinant Human ACE-2 Protein (933-ZN), Recombinant Human CA125/MUC16 Protein (5609-MU), Recombinant Human MUC-1 Fc Chimera Protein (10332-MU), Recombinant Human CD19 Fc Chimera Protein (9269-CD), Recombinant Human PD-1 Fc Chimera Protein (1086-PD) and Recombinant Human IL-6R alpha Protein (227-SR) were from Bio-Techne®. Fetal bovine fetuin was from Sigma Aldrich.
ST6Gal1 is used for N-glycan labeling. If the sample protein is partially sialylated, Neuraminidase may be omitted from the labeling reaction.
Prepare 1X Glycan Labeling Assay Buffer by diluting the 10X Glycan Labeling Assay Buffer with deionized or distilled water.
- N-glycan Labeling Reaction
- Add 1-5 µg of a sample protein, 1 µL CMP-Cy3-Neu5Ac, 1 µL ST6Gal1, and 1 µL Neuraminidase to a test tube.
- For assay control, assemble a new reaction by replacing the sample protein with 1 µL ACE-2 Protein.
- Add 1X Assay Buffer to a total reaction volume of 20 µL.
- Incubate at 37 °C for 60 minutes.
- N-glycan labeling is complete, proceed to downstream applications such as SDS-PAGE, Cell-based Assay, or PNGase F N-glycan Releasing Kit (Catalog # EA006).
- SDS-PAGE Gel Separation and Imaging (Optional)
- For a gel control, add 1 µL of Cy3-Neu5Ac Labeled ACE-2 Protein and 19 µL 1X Assay Buffer to a new test tube.
- Add 6X SDS Gel Loading Buffer to all the above tubes.
- Heat the tubes at 95 oC for 2 minutes.
- Load reaction to SDS-PAGE Gel.
- Run the gel until the dye front reaches the end of the gel.
- Perform fluorescent imaging first.
- Perform regular protein imaging via Silver, TCE or Coomassie® Blue staining.
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