Bio-Rad Bio-Plex® Assay Builder - Manual (Page 13)

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Expired beads, detection antibody,
and/or streptavidin-PE used
Incorrect incubation temperature
used during incubation steps
Incubation time insufficient
Low Bead Count
Cell debris in lysate not
cleared
Resuspension buffer not used
after streptavidin-PE incubation
Vacuum setting too high
Filter plate not shaken enough
before each incubation step
and prior to data acquisition
Reader clogged
High Coefficient of Variation (CV)
Plate sealer reused
Buffer not completely filtered
from wells
Possible Causes Possible Solutions
Use new or unexpired components
Incubations should be at room
temperature (20–22ºC)
Adhere to the recommended
incubation times
Remove the cellular debris by
centrifugation at 4,500 g for 20 min at
4ºC. Avoid disturbing the pellet while
collecting the supernatant
Rinse the beads 3 times with
resuspension buffer after the
streptavidin-PE incubation step
This results in bead loss. Calibrate the
vacuum apparatus as specified
Shake the filter plate at 1,100 rpm for
30 sec before each incubation and
immediately before acquiring data
Refer to the troubleshooting guide in the
Bio-Plex hardware instruction manual
This could result in contamination. Use
a new sheet of sealing tape for each
incubation
Be sure that the wells are filtered
completely and that no residual volume
remains
Section 8
Troubleshooting
This troubleshooting section addresses problems that may be encountered
with Bio-Plex phosphoprotein or total target assays. If the problems listed
below are encountered, review the possible causes and solutions provided.
This will assist in resolving problems directly related to the assay. Use the
Bio-Plex validation kit to validate all the key functions of the array reader
and assist in determining whether or not the array reader is functioning
properly.
Possible Causes Possible Solutions
Filter Plate Leakage
Vacuum setting too high
Low Signal (Good Signal From
Lysates Provided with the Assays but
Weak or No Signal From Experiment
Lysates)
Protein concentration in
lysate too low or too high
Low Signal (Weak or No Signal From
Lysates Provided with the Assays and
Experiment Lysates)
Detection antibody and/or
streptavidin-PE diluted incorrectly
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This could result in tearing of the filter.
Confirm that the vacuum pressure is
set as specified in the vacuum
calibration procedure section. Also
refer to the Vacuum Manifold Setup
in the Bio-Plex suspension array
system hardware instruction manual.
Use the recommended filter plate
vacuum apparatus
Verify the protein concentration in the
cell lysate samples. Adjust the
amount of lysing solution used in the
lysate preparation to achieve an
optimal protein concentration of
200–900 µg/ml prior to adding an
equal part of assay buffer
Check the calculations and be careful
to add the correct volumes for dilution