Gly-X™ N-Glycan Rapid Release and Labeling with 2-AB Express™ Kit (96-ct) [GX96-2AB]

Price:

Description

The Gly-X™ N-Glycan Rapid Release and Labeling with 2-AB Express™ kit utilizes a novel in-solution enzymatic protein deglycosylation followed by Express labeling of released N-glycans with 2-AB dye. With deglycosylation carried out in solution, the method is simple, rapid and suitable for automation. After a simple clean-up step, the 2-AB-labeled glycan samples are ready for analysis by UHPLC, LC-MS, MS/MS and other methods. 

Other benefits include:

    • Reductive amination 2-AB Express labeling chemistry without dry down
    • 5-minute PNGase F digestion provides unbiased N-glycan release
    • Flexible, high-throughput format: process 1 to 96 samples
    • Optimized cleanup removes excess free dye, protein, and other interfering compounds.

    Contains:

      • GX96-100 Gly-X Deglycosylation Module (96-ct)
      • GX96-401 Gly-X 2-AB Express Labeling Module (96-ct)
      • GX96-402 Gly-X 2-AB Express Clean-up Module (96-ct)

      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)
        • ProZyme Vacuum Manifold Spacer (GX100, or GX400 Gly-X 2-AB Starter Kit - included free of charge with first Gly-X order, please contact us for details)
        • Acetonitrile (ACN), MS-grade (Fisher A955-4 recommended
        • DI Water

        Size: 1 kit (96-ct)

        Product Code: GX96-2AB

        Tech Info

        GX96-2AB Gly-X with 2-AB Express User Manual

        FAQs

        Q. Do you have a list of masses for adduct ions of labeled N-glycans seen by MS in positive and negative mode?

        A. Yes, we have a downloadable Excel file listing masses of adduct ions of unlabeled and labeled glycans. The positive mode tab shows glycans labeled by 2-AB, InstantAB, 2-AA, Procainamide and InstantPC. The negative mode tab shows glycans labeled by 2-AB, InstantAB, 2-AA, Procainamide and APTS.

        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. 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: Can I use Eppendorf microtubes for the Denaturation and Digestion Steps, rather than a PCR plate?

        A: Yes, leave the tubes open for the heating steps, and ensure that material does not get in the lid during mixing.

        Q: Is it possible to use a plate shaker/heater such as the Eppendorf Thermo Mixer C formixing and heating during the Denaturation and Digestion protocols?

        A: Yes, this is possible. Mix rate should be high enough to vortex material in wells.

        Q: What is the Finishing Reagent for prior to the 2-AB labeling step?

        A: The Finishing Reagent is a weak acid, and drives conversion of the glycosylamine (-NH2) to a glycan with a free reducing end (-OH) prior to 2-AB labeling by reductive amination.

        Q: For the 2-AB labeling step, the heat block is at 75°C. Is this too high for 2-AB labeling?

        A: There is some inefficiency of heat transfer between the block and the plate, so the in-plate temperature is ~65°C. If performing the incubation in an oven, the temperature should be set to 65°C.

         

        Safety

        Product Safety Documentation for GX96-IPC:

        Product/Part No. Description
        WS0343 Gly-X N-Glycanase (PNGase F)
        WS0344 Gly-X Digestion Buffer
        WS0345 Gly-X Denaturant
        WS0359 Gly-X 2-AB Dye
        WS0360 Gly-X 2-AB Reductant
        WS0361 Gly-X 2-AB Catalyst
        WS0362 Gly-X 2-AB Finishing Reagent
        Publications

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

        Certificates of Analysis

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