Glossary of Terms
175
P/N CLS151164 Rev. B
LabChip GX Touch/GXII Touch User Manual
PerkinElmer, Inc.
Protein Assay Analysis
Protein assays utilize a chemistry that generates an extra set of
peaks (system peaks) just above the lower marker that should not
be included in the analysis. These system peaks must be identified
and excluded from further quantitative analysis. For ladder analysis,
peaks occurring after the lower marker less than the “Ladder Ratio”
analysis setting, are tagged system peaks to avoid confusion with
legitimate ladder peaks.
Protein samples contain a single lower marker. Alignment to a
single marker does not provide enough constraints to align large
proteins, so data is aligned to two ladders, one sipped just before
the sample wells and another sipped just after the sample wells.
The second ladder is called the bracket ladder. Samples are scaled
so the sample’s lower marker is nearly aligned with both ladder
lower markers. The scaling is weighted by the proximity in sip time
to each ladder. The sample sipped closest to the primary ladder is
scaled to align more closely to the primary ladder and the sample
sipped just before the bracket ladder is scaled to align most closely
to the bracket ladder. Then each sample is shifted in time so the
sample's lower marker aligns exactly with the primary ladder lower
marker.
After alignment, the size of the protein producing each peak is
calculated from the aligned peak time using a log (size) versus
1/(Time-T0) fit to the primarily ladder peaks of known size and
measured migration time. T0 is determined empirically as the time
offset which delivers the best straight line fit to the ladder data, The
value of T0 used for the fit is exported in the Well Table.
To determine sample peak concentration, the peak areas are first
corrected to compensate for the fact that the fluorescence
intensities are sampled at a constant time interval so slower moving
proteins spend more time under the detector than fast moving
proteins. The peak concentration is then calculated using the ladder
peak areas and concentration for the ladder. Analysis settings can
only be changed when reviewing LabChip GX Touch data in the
LabChip GX Reviewer software.
To quantitize the sample peak concentration based on a different
standard, the new standard must be added into each sample well at
a known concentration. The analysis settings provide a Sample
Peak Quantitation option using the peak area and concentration of
the User Standard instead of the ladder concentrations. Analysis
settings can only be changed when reviewing LabChip GX Touch
data in the LabChip GX Reviewer software.
