Bio-Rad Rat Diabetes Assays - Manual (Page 28)

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6. Wash the plate three times with 100 µl wash buffer.
7. Vortex the diluted (1x) detection antibodies gently for 5 sec.
Pour into a reagent reservoir and transfer 25 μl to each well of the
assay plate using a multichannel pipet.
8. Cover plate with sealing tape and protect from light with aluminum foil.
Incubate on shaker at 850 ± 50 rpm for 30 min at room temperature.
Prepare and Add Streptavidin-PE (SA-PE)
1. While the detection antibodies are incubating, use Table 18 to
calculate the volume of SA-PE (100x) and assay buffer needed.
Streptavidin-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 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 (100x stock) adjusted to a final
volume of 50 µl in assay buffer.
Table 18 shows an example calculation.
Table 18. Preparing 1x SA-PE from 100x stock (includes 25% excess volume).
# of Wells 100x SA-PE, µl Assay Buffer, µl Total Volume, µl
96 60 5,940 6,000