Bio-Rad Bio-Plex Pro™ Human Cancer Biomarker Assays - Manual (Page 22)

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Prepare and Add Streptavidin-PE (SA-PE)
1. While detection antibodies are incubating, use Table 11 or the Calculation
Worksheet on page 35 to calculate the volume of SA-PE and assay
buffer needed. SA-PE should be prepared 10 min before use.
2. Add the required volume of assay buffer to a 15 ml polypropylene tube.
3. Vortex the 100x stock of SA-PE for 5 sec at medium speed. Perform
a 30 sec spin to collect the entire volume at the bottom of the vial.
4. Dilute SA-PE to 1x by pipetting the required volume into the 15 ml
tube. Vortex and protect from light until ready to use.
Each well of the assay requires 0.5 μl of the 100x stock adjusted to a
final volume of 50 μl in assay buffer.
Table 11. Preparing 1x SA-PE from 100x stock (includes 25% excess volume).
5. After detection antibody incubation, slowly remove and discard
the sealing tape.
6. Wash the plate three times with 100 µl of wash buffer according
to the wash method of choice.
7. Vortex the diluted (1x) SA-PE at medium speed for 5 sec. Pour
into a reagent reservoir and transfer 50 µl to each well using a
multichannel pipet.
8. Cover with a new sheet of sealing tape and incubate in the dark for
10 min at room temperature with shaking at 850 ± 50 rpm.
9. After the streptavidin-PE incubation step, slowly remove and discard
the sealing tape.
# of Wells 100x SA-PE, µl Assay Buffer, µl Total Volume, µl
96 60 5,940 6,000
48 30 2,970 3,000