Gly-X™ Deglycosylation and 2-AB Express™ Labeling (96-ct) [GX96-401AB]

Price:

Description

The Gly-X™ Deglycosylation and 2-AB Express™ Labeling kit utilizes a novel in-solution enzymatic protein deglycosylation followed by Express on-matrix labeling of released N-glycans with 2-AB dye. With deglycosylation carried out in solution, the method is simple, rapid and suitable for automation. 

Other benefits include:

    • Reductive amination 2-AB labeling chemistry without dry down
    • 5-minute PNGase F digestion provides unbiased N-glycan release
    • Flexible, high-throughput format: process 1 to 96 samples

    Contains:

      • GX96-100 Gly-X Deglycosylation Module
      • GX96-401 Gly-X 2-AB Express Labeling Module

        Equipment and reagents provided by user:

          • Gly-X™ 2-AB Express™ Cleanup Module (96-ct) [GX96-402]
          • 96-well Thermocycler or two independent heat blocks
            • Thermocycler (Corning THERM-1001, 110V; THERM-1000, 230V)
          • Acetonitrile (ACN), MS-grade (Fisher A955-4 recommended)

          Size: 1 kit (96-ct)

          Product Code: GX96-401AB

          Tech Info

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

          FAQs

          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 GX96-401AB:

          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.