Bio-Rad Experion Protein Analysis Kits - Manual (Page 47)

Error Probable Cause Recommended Action
No peaks were detected
in some samples There is not enough sample in the wells Ensure that pipets are calibrated and that 6 µl solution has
been added to the wells
Samples were not prepared properly Review the sample preparation procedure detailed in
Chapter 3. The Pro260 ladder must be prepared with
reducing sample buffer even when samples are to be
separated under nonreducing conditions
Pro260 stain was photobleached Protect the Pro260 stain, gel-stain solution (GS), sample
buffer, and prepared and diluted samples from light
Prepare new GS; use a new tube of Pro260 stain if
necessary
Particulates are clogging the microchannels Use only high-quality, 0.2 µm-filtered water (such as
ReadyPrep
™ proteomics grade water, not autoclaved water)
Verify that the gel (G) and GS were properly filtered
If using samples that contain particulates, perform a quick
microcentrifuge spin of the prepared sample to pellet
particulates before loading
No peaks detected in any
samples Gel-stain solution (GS) is old or has been
photobleached Prepare fresh GS
Chip was not primed with the correct pressure
and time settings Check settings on the priming station and repeat the
analysis with a new chip
Particulates are clogging the microchannels Use only high-quality, 0.2 µm-filtered water (such as
ReadyPrep proteomics grade water, not autoclaved water)
Verify that the gel (G) and gel-stain solution (GS) were
properly filtered
If using samples, such as cell lysates, that contain
particulates, perform a quick microcentrifuge spin of the
prepared sample to pellet particulates before loading
Lower and/or upper marker
is missing Samples do not contain sample buffer or were
not prepared properly (if both are missing) Review the sample preparation procedure in Chapter 3
Late migration If only the upper marker is missing, refer to the
troubleshooting tips for late migration (below)
Electropherogram peaks are
much smaller than expected
(peaks are <50% of what is
expected) Too much stain in the gel-stain solution (GS);
sample cannot destain completely, and the
background is high; too much stain can
contribute to high background (>1,000 RFU)
and low sample and ladder peaks Prepare new GS using a new tube of stain. Ensure stain is
thawed completely before preparation
Not enough sample was added Ensure pipets are calibrated
Use pipets that accurately deliver volumes of 10 µl or less
Too much salt in the sample; salt ions compete
with the sample ions during injection Analyze a dilution series of the sample in water on
another chip. If the peak heights and calculated protein
concentrations of the diluted sample increase, the salt is too
high in the original sample. Determine the proper dilution to
minimize the salt effect
One or more components in the sample is
beyond the concentration range listed in the
compatibility table (Appendix C) Analyze a dilution series of the sample in water on another
chip. Adjust your sample preparation procedure as
necessary
Gel-stain solution (GS) is old or has been
photobleached Prepare fresh GS
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Experion Pro260 Analysis Kit