HT Protein Charge Variant Kit User Guide - PerkinElmer

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HT Protein Charge Variant Kit User Guide Why is the baseline noisy, the migration of my sample slow, and the resolution of the variants poor? If the HT Protein Express, the HT Low MW Protein, the HT Pico Protein, or the ProfilerPro Glycan Profiling assay was run on the instrument, then the electrodes must be cleaned before running the HT Protein Charge Variant assay. If the electrodes are not cleaned, then residual gel will contaminate the chip and adversely affect the performance. To fix this problem, remove the chip from the instrument, aspirate and rinse the wells of the chip as described in the Chip Preparation section, and add fresh Running Buffer. Next, replace the buffer tube with a new tube containing fresh Running Buffer. Then, thoroughly clean the electrodes of the instrument, using molecular biology-grade water and a swab. Place the chip back in the instrument and restart the run. _________________________________________________________________________________________ Caliper - a PerkinElmer Company Page 14 of 18 PN: CLS135474 Rev. 02 68 Elm Street Hopkinton, MA 01748-1668 1-877-LABCHIP (1-877-522-2447)
HT Protein Charge Variant Kit User Guide Frequently Asked Questions Kit Components 1. Is there a lower marker for this assay? No. The assay has been optimized to minimize variation in migration time (see Troubleshooting section for more details). Alignment to a lower marker should not be necessary. 2. Is there a charge or pI ladder for this assay? No. Similar to conventional CZE assays, this assay does not provide pI information. Sample Preparation 1. May other desalting methods be used, besides Zeba Desalting plates and columns? Yes, however Zeba Desalting plates and columns were used in the validation of the assay and are therefore preferred. 2. What happens to the dye when it is not used immediately after dilution with DMF? When left at room temperature for extended periods of time (hours), the dye mixture will degrade, causing inefficient labeling and resulting in low sample signal. 3. Can labeled samples be used on another day? We recommend that samples are analyzed immediately after labeling. However, labeled samples may be frozen and analyzed later. A loss in signal may result from this storage. 4. Can labeled samples be reconstituted in less or more than 60 µL of molecular-grade water? The 60 µL dilution gives the optimal signal and profile reproducibility. Other dilutions may be used; however, signal and reproducibility may be compromised. 5. Can the samples be labeled for more than 10 minutes, or less than 10 minutes? The recommended 10 minutes is the shortest labeling time that will give the specified reproducibility. Shorter labeling times will compromise reproducibility; longer labeling times are not likely to affect reproducibility. 6. What is the lowest sample concentration that I can use? The recommended range is 0.5 - 10 mg/mL. The reproducibility of % Relative Amount measurements may decrease for concentrations < 0.5 mg/mL, but detection below this limit is possible. 7. Can I label less than 24 samples? Yes. To label less than 24 samples, reduce the volume of Dye Concentrate and N,Ndimethylformamide proportionately. For example, to label 12 samples, add 2.5 µL Dye Concentrate to 72.5 µL N,N-dimethylformamide. 8. Can I analyze a sample with a pI outside of the specified range? Probably not. Lower pI mAbs may migrate too slowly and not elute before the end of the separation time, even for the 110s assay. Higher pI mAbs may not be sufficiently resolved, even at pH 7.2. 9. Can proteins other than monoclonal antibodies be tested using this assay? The HT Protein Charge Variant assay was developed using only monoclonal antibodies. However, it may be possible to resolve the variants of other proteins with pI in the range of 7 - 9.5. _________________________________________________________________________________________ Caliper - a PerkinElmer Company Page 15 of 18 PN: CLS135474 Rev. 02 68 Elm Street Hopkinton, MA 01748-1668 1-877-LABCHIP (1-877-522-2447)
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HT Protein Charge Variant Kit User Guide - PerkinElmer

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