N-Glycan Kit (RF Method, V1) (96 Well)

Product Overview

Typical characterization of N-glycan profiles involves cleaving glycan chains with PNGase F, labeling with the derivatization reagent of 2-AB, followed by analysis via high-performance liquid chromatography (HPLC). For the conventional 2-AB labeling workflow, enzymatic digestion duration and derivatization labeling time are the two primary rate-limiting steps. This rapid N-glycan analysis kit enables rapid enzymatic digestion, labeling and enrichment (higher fluorescence and mass spectrometry signals compared with 2-AB), with the entire sample pretreatment procedure taking less than 45 minutes.

Key Features

96-well plate compatible workflow

Designed for 96-well format processing, enabling parallel sample preparation and improved workflow efficiency in batch analysis.

Streamlined three-step process

Integrates enzymatic digestion, labeling, and SPE-based enrichment/purification into a simplified workflow suitable for high-throughput applications.

Rapid enzymatic digestion

PNGase F digestion can be completed in approximately 5 minutes at 50 °C, under recommended conditions.

Fast labeling reaction

Labeling reaction can be completed in approximately 5 minutes at room temperature.

Efficient SPE-based enrichment and purification

Solid-phase extraction provides a typical recovery rate of >90%, with a processing time of ≤10 minutes per sample. When combined with a 96-well plate and vacuum manifold system, up to 12 samples can be processed in approximately 1.5 hours under optimized conditions.

Direct compatibility with LC/LC-MS analysis

SPE-prepared samples can be directly used for LC or LC-MS analysis without additional purification steps.

Applications

High-throughput N-glycan profiling

Designed for parallel preparation and analysis of N-linked glycans from glycoproteins in 96-well format workflows.

Batch sample preparation for glycan analysis

Suitable for laboratories requiring efficient processing of multiple samples for LC or LC-MS-based glycomics studies.

LC/LC-MS-based glycan characterization

Applicable in liquid chromatography and mass spectrometry-based N-glycan analysis for research applications.

Performance Data

Performance Summary

No.	Performance	Test Protocol	Criteria		Results of Hzymes
1	Enzyme digestion efficiency	1. Treat samples of IgG1/IgG4/fusion protein with PNGase F according to IFU 2. Use CE-SDS to detect digestion efficiency.	Fully Digested		YES
2	Blank Control	1. Prepare IgG samples and blank solution according to the IFU 2. No interfering peak shall appear at the retention time of the peak in IgG sample for the blank solution	No interfering peaks in blank solutions		YES
3	Repeatability	1. Prepare 6 parallel samples of IgG 2. Calculate the CV of G0F area & G0F% of the 6 samples	G0F	Area CV ≤ 15%	2.2%
3	Repeatability		G0F	%Area CV ≤ 10%	1.1%
4	Intermediate Precision	1. Prepare 6 parallel samples of IgG by two persons separately 2. Calculate the CV of G0F area & G0F% of the 12 samples	G0F	Area CV ≤ 15%	4.5%/4.4%/4.7%
4	Intermediate Precision		G0F	%Area CV ≤ 10%	2.3%/2.1%/1.9%
5	Linearity	1. Prepare linear solutions of IgG (20%/40%/100%/200%/400%, from 7.5 μg to 160 μg) 2. Calculate the correlation coefficient R2 of sample concentration and G0F peak area	R² of Area and concentration of G0F: R² ≥ 0.990		0.997/0.999/0.999
6	Inter-Batch Variation	1. Take 3 batches of reagents, and prepare 3 samples with 3 replicates per batch independently 2. Calculate the CV of G0F	G0F	Area CV ≤ 15%	2.83%
6	Inter-Batch Variation		G0F	%Area CV ≤ 10%	0.29%

Inter-Batch Variation

Linearity

FAQ

How should protein amount be adjusted in the assay?

Do not change the sample addition volume of 20μL, as this may affect the performance of the reaction system. If adjustment is required, please modify the protein concentration instead of the loading volume.

What is the recommended linearity range of the kit?

The recommended linearity range is 15-120μgprotein, corresponding to a sample concentration of approximately 0.75-6 mg/mL.

Linearity may vary depending on sample type and can be further optimized if needed.

What is the recommended linearity gradient points for method setup?

Suggested calibration points include: 10, 20, 40, 80, and 120μgprotein. These can be adjusted according to experimental requirements or sample characteristics.

How should the labeling reagent be handled after reconstitution?

The labeling reagent is recommended for immediate use after dissolution. If aliquoting is necessary, it should be divided immediately after reconstitution and stored at -80 °C for up to 5-7 days.

Is precipitation normal after adding acetonitrile?

Yes. Slight precipitation after addition of acetonitrile is expected and is caused by phase incompatibility between aqueous and organic solvents. This is generally not indicative of assay failure under recommended conditions.

Can the recommended sample volume be adjusted?

The recommended sample volume is 200μL. It may be adjusted depending on sample properties and instrument requirements. Optimization is recommended based on specific experimental conditions.