Bio-Rad Experion Protein Analysis Kits - Manual (Page 49)

Error Probable Cause Recommended Action
Protein quantitation is
incorrect Upper and lower markers were not properly
assigned by the software Check that both markers were properly assigned. Manually
select the marker(s), if necessary (follow the instructions in
Section 6.3, Manually Setting a Marker)
Pipetting and/or dilution errors occurred during
sample preparation or chip loading Ensure your calculations and dilutions are correct
Ensure that pipets are calibrated, and use pipets that
accurately deliver volumes of 10 µl or less
Do not modify the sample preparation and loading protocols
described in this manual
Ensure that the total protein concentration of your samples
(including any user-defined internal standards that may be
added) is within the linear dynamic range before proceeding
with the sample preparation instructions described in
Section 3.4; if total protein concentration is unknown, run a
Pro260 analysis of a dilution series of your sample
Review the essential practices described in Chapter 2
before initiating another analysis with a new chip
Samples are old or have not been stored
properly, and the proteins degraded Prepare fresh samples and try the analysis again
Samples were not denatured properly;
regardless of whether reducing or nonreducing
conditions are used, the Pro260 ladder and
samples must be heat-denatured for the assay
to run properly Heat the samples and the Pro260 ladder for 3–5 min at
95–100°C before loading them into the chip. Keep the
caps of the sample and ladder tubes closed during heating
to prevent loss of vapor and subsequent changes in
concentration. Briefly centrifuge the samples before opening
Samples do not have the same staining
properties as the upper marker Use a different internal standard or a calibration curve
(see Chapter 5, Protein Quantitation Methods, for more
information)
Ghost peaks or
contaminants appear in
electropherograms Electrodes are contaminated Follow the deep cleaning procedure described in Appendix
B and the software Help section (search term “electrodes”)
Use only 0.2 µm-filtered water (such as ReadyPrep
proteomics grade water) for diluting the protein samples and
Pro260 ladder; do not use autoclaved water
Upper or lower marker is
incorrectly identified Sometimes Experion software selects the
incorrect peak to correspond to the upper or
lower marker. Such an error affects alignment
and sizing Manually select the upper and lower markers by following
the instructions in Section 6.3
Portion of the
electropherogram is
incorrectly identified as a
peak Default integration settings are inapporpriate
for the sample Manually exclude a peak (see Section 6.4) or change the
peak finding parameters (see Section 6.5)
Virtual gel does not
resemble an SDS-PAGE
separation of the same
samples The gel view is a direct interpretation of
the electropherogram data. By default, the
fluorescence intensity of the bands in each
lane is scaled to the intensity/peak height
of the highest peak in that sample (scale to
local). Consequently, each gel lane may have
a different scale of fluorescence intensity. In
addition, Experion Pro260 analysis differs from
SDS-PAGE in several ways that may impact
separations Adjust scaling of the virtual gel to Scale To Global
(see Section 4.2). This also adjusts the scale on all the
electropherograms. Adjust the intensity bar to the right of
the virtual gel as needed
In an Experion Pro260 analysis, the separation of the 10 kD
and 260 kD markers is almost linear, whereas in SDS-PAGE
it is logarithmic. To mimic the appearance of an SDS-PAGE
separation, select Tools > Options > Advanced and select
the Gel Mobility Correction option
Glycoproteins are not
separating as expected Large and complex glycosolated proteins may
not migrate as expected with the Experion
Pro260 assay; however, their separation
characteristics will be reproducible For more information, refer to Bio-Rad bulletin 5453 or
contact Technical Support
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com 45
Experion Pro260 Analysis Kit