Glossary of Terms
176
P/N CLS151164 Rev. B
LabChip GX Touch/GXII Touch User Manual
PerkinElmer, Inc.
Protein Charge Variant Assay
Capillary Zone Electrophoresis (CZE) is an electrophoretic
separation technique used to evaluate the charge heterogeneity of
proteins in a sample. The LabChip GXII Touch performs a
microfluidic adaptation of this technique for the Protein Charge
Variant Assay. For Protein Charge Variant assays, the separation
channel does not contain a polymer gel because the analytes are
not separated by size.
Protein Charge Variant assays separate analytes based on
differences in their net charge: molecules with a higher net charge
migrate faster than those with a lower net charge. The relative
difference in migration speeds (and therefore the resolution)
between molecules of different pI is higher when the pH of the
running buffer is closer to the pIs of the molecules.
For the HT Protein Charge Variant assay, the pH of the running
buffer is less than the pIs of the variants, so molecules have a net
positive charge. Variants that are more basic (have a higher pI) than
others appear earlier in electropherogram. The software can be
used to track expected variants, based on migration time, and to
determine the relative amount of each variant, based on peak area.
Protein Charge Variant assays do not use ladders or markers to
align the data. The size of a peak is not calculated, only the % Rel
amount of each peak. Protein Charge Variant assays and data files
do not include any ladder or marker parameters or options. Analysis
settings can only be changed when reviewing LabChip GX Touch
data in the LabChip GX Reviewer software.
RNA Assay Analysis
RNA analysis initially progresses similarly to DNA analysis except
that the baseline for RNA peaks is calculated using a spline-fit,
much like the baseline used for the Baseline Subtraction option.
However, this computed fit is not subtracted from the data; instead it
is used to determine the peak height and to limit the peak extends.
The RNA ladder is similar to the DNA ladder but does not contain
an upper marker. RNA sample data is aligned with the lower maker
and then the sample peaks are sized using point-to-point
interpolation between ladder peaks. The most prominent peaks
found within predefined size windows are identified as ribosomal
rRNA genes 5S, 18S and 28S (for eukaryote RNA), 16S and 23S
(for prokaryote RNA).
