Bio-Rad Bio-Plex® Assay Builder - Manual (Page 7)

Section 6
Assay Instructions
The following instructions apply to Bio-Plex phosphoprotein and total
target singleplex, custom-premixed, and mutliplex assays. Do not mix
phosphoprotein assays with its corresponding total target assays (e.g.
phospho-Akt and total Akt).
Plan Experiment
1.
Assign which wells of a 96-well plate will be used for each lysate
(see the example below). Keep in mind that the instrument reads
wells down the plate and not across. Consider assigning the wells
vertically. A pullout worksheet has been provided in this manual that
may be used as a reference during the different assay steps.
2. Determine the total number of wells that will be used in the assay.
Include a 25% excess (or add 2 wells for every 8 wells used) to
ensure that enough diluted coupled beads, detection antibodies,
and streptavidin-PE are prepared. Record these numbers on the
worksheet since they will be referenced throughout the assay.
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Example Plate
7. Add an equal volume of assay buffer to the lysate.
8. If the lysate is not tested immediately, store at –20ºC. The lysate is
stable for up to 5 freeze-thaw cycles.
Suggested protocol for lysate preparation of Histone H3 assay:
1. Follow steps 1–3 above.
4. Lyse the samples:
a) Immediately add the lysing solution to the cells. The amount of
lysing solution needed depends on the cell concentration in the
culture vessel (see table on the left).
b) Briefly sonicate (e.g., with a Sonifer 450 as follows:
Duty cycle = 40, Output = 1, Pulse sonicating = two 10 min
pulses with a 1 min break in between).
c) Agitate the cells. Transfer the cell lysate to a centrifuge tube and
rotate for 20 min at 4ºC.
d) Centrifuge the samples at 4,500 g for 20 min at 4ºC.
5. Collect the supernatant without disturbing the pellet.
6. Determine the lysate protein concentration. The protein concentration
should be 200–900 µg/ml. It may be necessary to test-lyse your
samples with different volumes of lysing solution to obtain the
specified protein concentration range.
7. Add equal volume of assay buffer to the lysate.
8. Freeze (overnight) at -20ºC and thaw before testing.
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