Bio-Rad Bio-Plex® Assay Builder User Manual
Page 9
Assay Procedure
Bring all buffers to room temperature. Avoid bubbles when pipetting.
1.
Wash the desired number of wells in a 96-well filter plate. If fewer
than 96 wells are required, cover the unused wells with sealing tape
for later use.
2.
Vortex the coupled beads (1x) for 5 sec at medium speed. Add
50 µl to each well and immediately vacuum-filter.
3.
Wash twice.
4.
Vortex the thawed lysates gently for 3 sec. Add 50 µl of lysate to
each well, changing the pipet tip after every volume transfer.
Incubate for 15–18 hr (or overnight).
13
Volume of Detection Antibodies (25x) in Each Well
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6
Example Detection Antibody Calculations
Calibrate Vacuum Apparatus
The vacuum apparatus must be calibrated at the beginning of the assay
to ensure an optimal bead yield. For more detailed instructions, refer to
the Bio-Plex suspension array system hardware instruction manual.
1.
Prewet all the wells of a 96-well filter plate with 100 µl of wash buffer.
2.
Place the filter plate on the vacuum apparatus and turn on the
vacuum to the maximum level.
3.
Press on the filter plate and note the time required to remove the
buffer from the wells by vacuum filtration. The evacuation time
should be 2–5 sec.
If the evacuation time is <2 sec, the pressure is too high. Open the
vacuum control valve slightly and repeat steps 1–3.
If the evacuation time is >5 sec, the pressure is too low. Close the
vacuum control valve slightly and repeat steps 1–3.
Assay Key
The following terms are repeated throughout the assay procedure. Refer
to these detailed instructions when wash, rinse, incubate, and
vacuum-filter are shown in bold.
12
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