Bio-Rad Bio-Plex® Assay Builder User Manual

Page 13

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

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