Gly-X™ N-Glycan Rapid Release and Labeling with InstantQ™ Kit (96-ct) [GX96-IQ]

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Description

The Gly-X™ N-Glycan Rapid Release and Labeling with InstantQ™ kit utilizes a novel in-solution enzymatic protein deglycosylation followed by rapid labeling of released N-glycans with InstantQ dye. After a simple clean-up step, the glycan samples are ready for analysis on the Gly-Q Glycan Analysis System. With deglycosylation and labeling carried out in solution, the method is simple, rapid and suitable for automation.
Other benefits include:

    • Instant fluoresent dye optimized for high sensitivity detection
    • Flexible, high-throughput format: process 1 to 96 samples
    • Optimized cleanup removes excess free dye, protein, and other interfering compounds.

    Contains:

      • GX96-300 Gly-X Deglycosylation Module for InstantQ
      • GX96-301 Gly-X InstantQ Labeling Module
      • GX96-302 Gly-X InstantQ Clean-up Module
      • GKSQ-505 Gly-Q Alignment Standards Set (GKSQ-500 & GKSQ-503)

      Equipment and reagents provided by user:

        • 96-well Thermocycler or two independent heat blocks, set for 90 °C and 50 °C
          • Thermocycler (Corning THERM-1001, 110V; THERM-1000, 230V)
          • Note: Two GlykoPrep Heaters, WS0271, can be fitted with VWR 13259-260 Modular Heating Blocks
        • Vacuum manifold (Millipore MSVMHTS00-HTS)
        • Vacuum pump (Millipore WP6211560, 110 V; WP6122050, 220V)
        • Formic acid, MS-grade (Fisher A117-50 recommended)
        • Acetonitrile (ACN), MS-grade (Fisher A955-4 recommended)

        Size: 1 kit (96-ct)

        Product Code: GX96-IQ

        Specifications

        Gly-X with InstantQ Kit

        Sample prep time 96 samples in 1 hour
        Protein load range 1-40 µg
        Label Proprietary instant label
        Deglycosylation efficiency >99%
        Glycan recovery >85%
        Sialylated species >95% retained
        Precision <5% CV
        Sample stability 4°C for up to 5 days
        Sample stability -20°C for up to 3 months
        Kit configuration Modular
        Tech Info

        Gly-X with InstantQ Dye User Manual 

        Gly-Q Glycan Analysis System Product Guide 

        Publications

        An Integrated Solution for High-throughput, User-friendly Glycoanalysis Using Rapid Separation by Capillary Electrophoresis

         

        Ordering Info

        Kits and Modules

         Product Code

        Description

        GX96-IQ Gly-X with InstantQ Kit (96-ct)
        GX96-302IQ Gly-X with InstantQ Deglycosylation and Labeling Module Set (96-ct)
        GX24-302IQ Gly-X with InstantQ Deglycosylation and Labeling Module Set (24-ct)
        GX96-301 Gly-X InstantQ Labeling Module
        GX96-302 Gly-X InstantQ Cleanup Module
        G5524-60010 KIT AssayMAP PA50 protein A affinity purification kit (96-ct)
        GQ103 Gly-Q Cartridge Module

        Standards and Controls

         Product Code

        Description

        GKSQ-500 Gly-Q Migration Standards (upper and lower)
        GKSQ-503 Gly-Q GU Ladder
        GKSQ-505 Gly-Q Alignment Standards Set (GKSQ-500 & GKSQ-503)
        GKSQ-005 Human IgG N-Glycan Library
        GKSQ-009 InstantQ Labeled RNase B Library
        GKSQ-301 InstantQ Labeled G0
        GKSQ-302 InstantQ Labeled G0F
        GKSQ-317 InstantQ Labeled G1
        GKSQ-316 InstantQ Labeled G1F
        GKSQ-304 InstantQ Labeled G2
        GKSQ-305 InstantQ Labeled G2F
        GKSQ-315 InstantQ Labeled A1F
        GKSQ-312 InstantQ Labeled A2
        GKSQ-313 InstantQ Labeled A2F
        GKSQ-103 InstantQ Labeled Man5
        GKSQ-104 InstantQ Labeled Man6
        GKSQ-105 InstantQ Labeled Man7
        GKSQ-106 InstantQ Labeled Man8
        GKSQ-107 InstantQ Labeled Man9

         

        FAQs

        Q: When do I need to use the Gly-X Blocker with my deglycosylation reaction? What does the Blocker do?

        A: The Gly-X Blocker is optional. It is not required for most biotherapeutics e.g. IgGs, Fc fusion proteins. However, if you observe an uneven/rounded baseline during analysis we suggest running the protocol with and without the Blocker to test whether it is required. Potential scenarios that may require the Blocker are:

        a) if your glycoprotein is <50 kDa;

        b) if the glycoprotein contains a large proportion of basic amino acids;

        c) if your sample is a complex matrix such as a biofluid.

        The Blocker ensures that labeled protein does not interfere with N- glycan analysis by blocking free amine groups on the glycoprotein before they can be labeled with InstantQ dye.

        Using the blocker may cause the reaction to turn yellow, particularly when there is a lot of protein material. This is caused by Maillard reaction products and is not a cause for concern. Please contact ProZyme for further details.

        Q. Why should I use 0.1% formic acid to elute samples from Protein A ready for Gly-X with InstantQ, rather than a low pH glycine solution?

        A. Glycine has a primary amine which will react with the InstantQ label, so we suggest that 0.1% formic acid is used as a protein eluent. Alternatively, glycine may be used but samples must be desalted before deglycosylation.

        Q. Can I use ProZyme AssayMAP Protein A cartridges to purify proteins ready for Gly-X with InstantQ, and elute with 0.1% formic acid?

        A. Yes. AssayMAP PA50 Protein A Cartridges are available from ProZyme in 24-count (G5524-60001) and 96-count (G5524-60010) kits, and 0.1% formic acid may be used as an eluent.

        Q. What is the lower limit of glycoprotein I can use with the Gly-X/InstantQ protocol?

        A. Each laboratory will need to establish the lower limit for each specific protein. 0.125 mg/ml can be used as a starting point.

        Q: Can I use more than the recommended upper limit of 40 µg protein per reaction?

        A: Maybe, although it will depend on the protein. When using >40 µg protein the user should check that relative percent area data maintains linearity.

        Q: Can I run glycans labeled with InstantQ dye on other CE systems?

        A: The LIF detector typically used on the Beckman PA800-plus for APTS glycans is not compatible with the InstantQ Dye excitation wavelength. Suitability on other systems may be determined by the user.

        Safety

        Product Safety Documentation for GX96-IQ:

        Product/Part No. Description
        WS0343 Gly-X N-Glycanase (PNGase F) 
        WS0344 Gly-X Digestion Buffer
        WS0345 Gly-X Denaturant
        WS0349 InstantQ Dye
        WS0260-GP InstantQ Dye Solvent
        WS0354 InstantQ Activation Reagent
        Certificates of Analysis

        This kit contains multiple modules, each with their own Certificate of Analysis, which may be searched for & accessed here.


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