Deglycosylate with GKE-5006, or GKE-5020 (5020 at higher concentration). Use around 100ug per sample for a mAb, which has 2-3% N-glycosylation by weight (more or less will also work). Many people like to use native conditions for mAb deglycosylation as it can come out of solution in denaturing conditions.
If the customer is potentially going to buy a lot of enzyme, let us know, maybe there is the opportunity to work with them.
For cleaning after N-Glycanase (PNGase F) reaction I’d suggest ethanol precipitation, or the R cartridge. Glycan cleanup by ethanol precipitation after digestion & prior to 2-AB labeling has been used by many labs for many years.
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For 2-AB labeling - dry down sample in a SpeedVac then use GKK-804. Better kit than the older GKK-404. The two kits are interchangeable (see attached memo).
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For cleanup after labeling, S cartridge (most common) or H:
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None of these use a vacuum manifold. The ones we offer that do, are G and S-plus cartridges.
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For analysis, HPLC or UPLC is fine. HPLC will just take longer! Most people separate glycans using HILIC columns, Waters BEH glycan column is most popular but Agilent also have the advanceBio glycan mapping column. You can run the UPLC columns under higher pressure for faster/better separation. We run Waters H Class and I Class UPLCs at ProZyme.