N-Glycan Kit (RF Method, V2) (96 Well)

Product Overview

Typical characterization of N-glycan profiles involves cleaving glycan chains with PNGase F, labeling with the derivatization reagentof2-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 45minutes.

Key Features

96-well plate compatible high-throughput workflow

Designed for 96-well format operation, enabling parallel processing and improved efficiency in batch N-glycan sample preparation.

Integrated three-step workflow

Combines enzymatic digestion, labeling, and SPE-based enrichment/purification into a streamlined workflow suitable for routine and high-throughput analysis.

Rapid enzymatic digestion

PNGase F digestion can be completed in approximately 5 minutes at 50 °C, under non-denaturing or mild denaturing conditions as specified in the protocol.

Fast labeling reaction

2-AB derivatization labeling can be completed in approximately 5 minutes at room temperature under recommended conditions.

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

Samples prepared using the SPE workflow can be directly analyzed by LC or LC-MS without additional purification steps.

Applications

High-throughput N-glycan profiling

Designed for rapid preparation and analysis of N-linked glycans from glycoproteins in 96-well format workflows, suitable for batch sample processing and routine glycomics studies.

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	Fully Digested		YES
1	Enzyme digestion efficiency	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	No interfering peaks in blank solutions		YES
2	Blank Control	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	G0F	Area CV≤15%	2.20%
3	Repeatability	2. Calculate the CV of G0F area & G0F% of the 6 samples	G0F	%Area CV ≤ 10%	1.10%
4	Intermediate Precision	1. Prepare 6 parallel samples of IgG by two persons separately	G0F	Area CV ≤ 15%	4.5%/4.4%/4.7%
4	Intermediate Precision	2. Calculate the CV of G0F area & G0F% of the 12 samples	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)	R2 of Area and concentration of G0F: R²≥0.990		0.997/0.999/0.999
5	Linearity	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	G0F	Area CV ≤ 15%	2.83%
6	Inter-Batch Variation	2. Calculate the CV of G0F area & G0F% between batches respectively	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.

The linearity range may vary depending on sample type and experimental conditions, and can be further optimized if needed.

What are the suggested linearity gradient points?

Recommended calibration points include 10, 20, 40, 80, and 120μgprotein. These can be adjusted based on specific experimental requirements.

How should the labeling reagent be handled after reconstitution?

The labeling reagent is recommended for immediate use after dissolution. If aliquoting is required, 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 adding acetonitrile is normal and is caused by phase incompatibility between aqueous and organic solvents. This does not typically affect assay performance under recommended conditions.

Can the recommended sample volume be adjusted?

The recommended sample volume is 200μL. It can be adjusted depending on sample characteristics and instrument conditions. Optimization is recommended based on specific experimental setup.