ProfilerPro Glycan Profiling User Guide - PerkinElmer

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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)
ProfilerPro Glycan Profiling Kit, Ver 2, User GuideNo sample peaks (ladder is OK)There are several possible causes:1. The sipper is not reaching the sample due to low sample volume in the well plate, or wrong plate typeselected.2. The samples did not get labeled completely due to incomplete drying.3. If the missing sample peaks occurred only in a few wells of the plate, check those wells for air bubbles.4. Debris from the sample or sample prep is clogging the sipper.What to do:1. Make sure you have selected the Plate Type “96-Deep Well PCR with Adapter.” Verify the samplevolume is at least 100μL.2. Repeat the sample preparation procedures allowing the drying reaction to extend beyond 2 hours until thesamples are completely dry.3. Manually insert a larger volume pipette tip (~100µL) into the sample well and dislodge the bubble. Rerunthese sample wells.4. If you suspect there may be debris in your samples, spin the sample plate down in a centrifuge. Unclogthe sipper by repriming the chip. See the section entitled “LabChip Kit Essential Practices” forinstructions on how to reprime the chip.No sample or Ladder Peaks (Marker is OK)The sipper may be clogged. To fix this reprime the chip. Stop the run, press the “Chip” button on the instrumentto eject the chip cartridge, reinsert the chip by pushing the cartridge back into the instrument, and restart the run.Caliper Life Sciences Page 13 of 21 PN: 45070968 Elm StreetHopkinton, MA 01748-16681-877-LABCHIP (522-2447)
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Page 3 and 4: Kit ContentsThe ProfilerPro Glycan
Page 7 and 8: Starting the LabChip GX1. Start the
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ProfilerPro Glycan Profiling User Guide - PerkinElmer

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