Bio-Rad Human MMP and TIMP Assays - Manual (Page 23)

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7. Vortex the diluted (1x) detection antibodies at medium speed for
5 sec. Transfer into a reagent reservoir and dispense 25 µl to each
well of the assay plate using a multichannel pipet.
8. Cover plate with a new sheet of sealing tape and protect from light with
aluminum foil. Incubate on shaker at 850 ± 50 rpm for 30 min at RT.
Prepare and Add Streptavidin-PE (SA-PE)
1. While detection antibodies are incubating, use Table 11 or the
Calculation Worksheet on pages 35–36 to calculate the volume of
SA-PE and assay buffer needed to prepare a 1x stock. 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 tube.
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 per well.
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.
# of Wells 100x SA-PE, µl Assay Buffer, µl Total Volume, µl
96 60 5,940 6,000
48 30 2,970 3,000