Gly-X™ N-Glycan Rapid Release and Labeling with InstantPC™ Kit (24-ct) [GX24-IPC]

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Description

The Gly-X™ N-Glycan Rapid Release and Labeling with InstantPC™ kit utilizes a novel in-solution enzymatic protein deglycosylation followed by rapid labeling of released N-glycans with InstantPC dye. After a simple clean-up step, the glycan samples are ready for analysis by UHPLC, LC-MS, MS/MS and other methods. With deglycosylation and labeling carried out in solution, the method is simple, rapid and suitable for automation. The InstantPC dye delivers unmatched fluorescent brightness and MS signal, which enables a single labeling method to be deployed across different glycan analysis workflows.

Sample prep time: ~1 hour.

Other benefits include:

    • Small molecule size for improved chromatographic peak resolution
    • Flexible, high-throughput format: process 1 to 24 samples
    • Optimized cleanup removes excess free dye, protein, and other interfering compounds
    • Reconstituted InstantPC dye may be stored frozen (-20°C) for up to 1 month

    Contains:

      • GX24-100 Gly-X Deglycosylation Module
      • GX24-101 Gly-X InstantPC Labeling Module
      • GX96-102 Gly-X InstantPC Clean-up Module 
        • Note: The 24-count kit includes a 96-count cleanup plate. This plate can be stored at room temperature for future use. Gly-X InstantPC digestion and labeling reagents (GX24-201PC) are available separately for use with existing cleanup plates. 

    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; Welch WOB-L Pump 2522)
      • Formic acid, MS-grade (Fisher A117-50 recommended)
      • Acetonitrile (ACN), MS-grade (Fisher A955-4 recommended)

      Size: 1 kit (24-ct)

      Product Code: GX24-IPC

      Tech Info

      GX96-IPC Gly-X with InstantPC User Manual

      Gly-X with InstantPC Product Guide

      Gly-X InstantDye Automation Product Guide  

       

      FAQs

      Q. What is the most common adduct seen in MS analysis of InstantPC-labeled glycans?

      A. In positive mode MS, most biantennary InstantPC-N-glycans will give [M+2H]+2, larger sialylated will be majority [M+3H]+3.

      Q. You suggest 100 mM ammonium formate for the 5-minute HILIC separation, 50 mM ammonium formate for the 60-minute separations. Is this correct?

      A. For fast separations we stick with 100mM buffer, for MS in conjunction with longer separations we use 50mM ammonium formate.

      Q. Why is PBS not recommended as a diluent for glycoprotein samples?

      A. PBS is compatible with Gly-X InstantPC kit, however, use of PBS for sample dilution may result in artifact peaks. These peaks elute early in the separation, near the free dye peak and should not interfere with N-glycan analysis. For optimal performance, dilution of samples with water is preferred.

      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. You would have to test to ensure that labeled glycans from > 40 µg of your protein can be successfully loaded onto the Gly-X Cleanup Matrix without blockage. 

      Q. My samples are not loading completely onto the Gly-X Cleanup Matrix (Load Step 9, page 9). What is happening?

      A. This may be caused by the nature of your protein sample, or by matrix effects caused by the composition of your formulation buffer. This can be addressed by using less protein per reaction, or by buffer exchanging your protein prior to starting the Gly-X protocol.

      Q. Why can I not use ACN to dilute the samples prior to HILIC injection? And can I use something other than DMF for the dilution?

      A. If the InstantPC-labeled glycans in Gly-X Eluent are diluted with ACN alone, this may cause the sialylated glycans to precipitate. We have not tested alternatives to DMF for sample dilution.

      Safety

      Product Safety Documentation for GX24-IPC:

      Product/Part No. Description
      WS0343-24 Gly-X N-Glycanase (PNGase F)
      WS0344 Gly-X Digestion Buffer
      WS0358 Gly-X Denaturant
      WS0338 InstantPC Dye
      WS0339 InstantPC Dye Solvent
      WS0333 InstantPC Eluent
      Publications

      Comparison of common fluorescent labels for liquid chromatography analysis of released N-linked glycans

      Development of a 5-Minute Deglycosylation Method and Instant Labeling Dye for High Throughtput N-Glycan Analysis by Mass Spectrometry

      Development of an Instant Glycan Dye for High Throughput Analysis by Mass Spectrometry

      Certificates of Analysis

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