Bio-Rad Experion Protein Analysis Kits - Manual (Page 48)

Error Probable Cause Recommended Action
Reagents, specifically the Pro260 stain, were
not brought to room temperature prior to use; if
stain is not equilibrated to room temperature, it
will be more concentrated when it is added to
the gel, which may result in significantly lower
protein peaks and areas Repeat the analysis with a new chip, new tube of stain, and
properly equilibrated reagents
Gel (G) and gel-stain solution (GS) were
mislabeled or used improperly. The Pro260
chip contains specific wells for G and GS:
GS wells carry the sample through the
microchannels and supply the dye for analysis,
and the G well is used for destaining prior
to detection. If G and GS are added to the
incorrect wells, peaks can be small, crowded,
or missing in electropherograms Ensure chip was primed with GS
Ensure G was used in the G well and GS was used in all
wells marked GS
Peaks are small, broad, or
missing in some samples,
but present in other samples
on the chip Pro260 ladder and samples were not
completely denatured Heat samples and Pro260 ladder for 3–5 min at 95–100°C
before loading
Contaminants are present Check chemical compatibility charts (Appendix C) and
prepare samples in a different extraction buffer or dilute
them in water, if necessary
Do not use autoclaved water for diluting protein samples or
ladder. Use only high-quality, 0.2 µm-filtered water (such as
ReadyPrep proteomics grade water)
Late migration (peaks
broaden and are delayed
over the course of the run) Turn analysis off (see Section 6.8, Turning Analysis Off, for
more information). Late migration is indicated if there is a
drift in the migration of the lower marker across the chip and
the upper marker migrates later and runs off the top of the
gel view. Refer to the troubleshooting tips that follow
Sample pH is lower than 5 Adjust pH of the sample to 5 or above
Air bubbles are interfering with electrical
contact in one or more of the wells Stop the run, remove the chip, and use a clean pipet tip to
remove or dislodge the bubbles, or remove and replace the
solution in affected wells
When pipetting, insert the tip vertically and to the bottom of
the well. Dispense liquids slowly. Do not expel air at the end
of the pipetting step. Dispense only to the first stop
Peaks migrating faster than
usual; electropherogram
appears compressed Electrophoresis station temperature is
inappropriate or fluctuating during the run
(should be 30–35°C); there is no cooling
unit in the electrophoresis station, so if the
temperature changes during the course of a
run, the samples will exhibit different separation
characteristics Ensure that the temperature of the room is appropriate and
stable, and place the electrophoresis station away from all
heat sources, such as windows or ovens
Molecular mass (sizing) is
incorrect Pro260 ladder peaks and upper and lower
markers were improperly assigned by the
software Exclude peaks (follow the instructions in Section 6.4,
Excluding a Peak From Analysis) or manually assign markers
(follow the instructions in Section 6.3, Manually Setting a
Marker), if necessary
Sizing should be within 10% of the size seen by SDS-PAGE,
except for proteins with high levels of posttranslational
modification, or highly acidic or basic proteins
44 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
Experion Automated Electrophoresis System