Product Overview
The N-Glycan Kit 2.0 (single column) provides a complete workflow for the fast enzymatic release and rapid labeling of N-glycans. The protocol is validated using monoclonal antibodies and has also been tested on a wide range of other N-linked glycoproteins, and users are advised to confirm enzymatic release for their own sample type. The kit is organized into four modules: deglycosylation, labeling, clean-up, and collection. In the deglycosylation step, the supplied PNGase F buffer is added directly to the sample, with no separate surfactant reagent to dissolve and prepare beforehand. The sample is denatured at 90°C for 3 minutes and then treated with PNGase F at 50°C for 5 minutes. The typical protocol uses 20 μL of IgG at 2 mg/mL, and the concentration can be adjusted to the sample and the instrument. Released glycans are then labeled with the kit’s MS-active labeling reagent at room temperature in 5 minutes. In total, the incubation steps take roughly 20 minutes.
The clean-up module uses a single SPE column format. Labeled glycans are conditioned, equilibrated, loaded, washed, and eluted, which removes excess labeling reagent and reaction by-products before analysis. Purified glycans are analyzed by HILIC-FLR(-MS) on a Glycan BEH Amide column (130 Å, 1.7 μm, 2.1 × 150 mm) with fluorescence detection at Ex 265 / Em 425 nm. The validated 55-minute gradient resolves a complete IgG N-glycan profile, including neutral, fucosylated, bisected, and sialylated species such as G0, G0F, G1F, G2F, G1FB, G2FB, G1FS1, G2FS1, and G2FS2. Samples that contain interfering buffer components can be prepared with the buffer-exchange procedure described for this kit, which uses a 10 kD ultrafiltration tube.
Components
| Module | Reagent | 24 T |
| Deglycosylation Module | IgG | 1.2 mg |
| PNGase F | 45 µL | |
| PNGase F buffer | 0.5 mL | |
| Labeling Module | MS reagent powder | 8.2 mg×3 |
| Anhydrous DMF | 1 mL | |
| Clean-up Module | SPE column | 8 pcs/pk×3 |
| Elution Buffer | 5 mL | |
| Sample Diluent | 6 mL | |
| Collection Module | 8-tube Strips (200μL) | 100 pieces |
| 8-cap Strips | 100 pieces | |
| Waste tray | 1 pieces |
Product Features
- Simplified, fast deglycosylation: The supplied PNGase F buffer is used directly, so there is no separate surfactant reagent to prepare. Denaturation (3 min at 90°C) and enzymatic release (5 min at 50°C) are completed within minutes.
- Rapid room-temperature labeling and single-column clean-up: Glycans are labeled in 5 minutes at room temperature, and a single SPE column removes excess reagent and by-products without multiple column exchanges.
- Validated HILIC-FLR(-MS) profiling of IgG glycans: The chromatographic method is provided with the kit and resolves a complete IgG N-glycan profile, including neutral, fucosylated, bisected, and sialylated glycans, with fluorescence quantitation and MS confirmation.
FAQ
Q1: How does the N-Glycan Kit 2.0 differ from the original N-Glycan Kit (single column)?
Both kits follow the same overall workflow: PNGase F release, rapid labeling, SPE clean-up, and HILIC-FLR(-MS) analysis. In the 2.0 kit, the supplied PNGase F buffer is used directly in the deglycosylation step, so there is no separate surfactant reagent to dissolve and prepare before use. The 2.0 kit also comes with guidance for preparing samples that contain interfering buffer components.
Q2: What if my sample is in a buffer that contains citrate, acetate, phosphate, Tris, or SDS?
If the sample contains citrate, acetate, phosphate, Tris, or SDS, or if its pH is lower than 4 or higher than 9, and slight precipitation occurs during use that affects the results, solution replacement is recommended. The kit documentation describes a buffer-exchange procedure using a 10 kD ultrafiltration tube, in which the sample is washed with ultrapure water by centrifugation and then transferred. Protein concentration can be measured afterward and adjusted to a suitable level for N-glycan analysis. Centrifugation times and speeds may need adjusting for your equipment, consumables, and sample.
Q3: What do I need besides the kit, and how long does the workflow take?
You need 0.85% NaCl, LC-MS grade acetonitrile, ultrapure water, formic acid, and 50 mM ammonium formate (pH 4.4), as well as a heating module or metal bath that holds 90°C and 50°C, a vortex mixer, an SPE manifold, and an LC system with a Glycan BEH Amide column and fluorescence detection. The incubation steps (denaturation, enzymatic release, and labeling, including cooling) add up to roughly 20 minutes, followed by SPE clean-up and a 55-minute chromatographic run. The published method parameters were set up on an ACQUITY RDa LC-MS system and can be adjusted for other equipment.
