ProfilerPro Glycan Profiling User Guide - PerkinElmer

ProfilerPro Glycan Profiling Kit, Ver 2, User GuideTroubleshootingHow to test for complete digestionCaliper recommends testing for complete deglycosylation by PNGase F using the HT Protein Express assay.Compare the digested sample (unlabeled) to the undigested sample. Follow the protocol for the HT ProteinExpress Kit for reduced conditions. (Note: It may be necessary to dilute the digested sample depending on thestarting concentration. A starting concentration of 2mg/mL or below is recommended for the HT Protein Expressassay.) Under reduced conditions you will see a shift from the heavy chain (HC) to the non-glycosylated heavychain (NGHC) for the digested sample.Blue: control (undigested), Red: Digested MAbWhat if digestion is incompletePossible causes:1. The samples were incompletely denatured.2. There was an insufficient amount of PNGase F in the reaction.What to do:1. Repeat the sample preparation being careful to spin down the Denaturing Plate. Small amounts ofdenaturing buffer attached to the plate seal may significantly decrease the amount or concentration ofreagents needed to sufficiently denature samples. Additionally verify that the samples are beingdenatured at 70C for 10 minutes.2. If the sample tested was on the high end of the Glycan assay concentration range, then dilute sampledown to 5mg/mL and repeat sample preparation. Some MAbs may be more difficult to deglycosylate thanothers. In this event a greater amount of PNGase F to MAb may be need to completely deglycosylate.3. Repeat the sample preparation being careful to spin down the PNGase F Plate. Small amounts ofdigestion buffer attached to the plate seal may significantly decrease the available amount of PNGase F.Verify that the digestion is incubated at 37C for 1 hour.No peaks are found in the Ladder (sample and marker peaks are OK)There may be too low of a ladder volume or no ladder volume at all in the Ladder Tube. Try adding more ladderto the Ladder Tube and restart the run. Recommended standard ladder volume is 120µL (minimum requiredvolume is 100µL).Caliper Life Sciences Page 12 of 21 PN: 45070968 Elm StreetHopkinton, MA 01748-16681-877-LABCHIP (522-2447)