Bio-Rad Human MMP and TIMP Assays - Manual (Page 21)

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4. Dilute detection antibodies to 1x by pipetting the required volume into
the 15 ml tube. Vortex.
Each well of the assay requires 1.25 μl of the 20x stock adjusted to a
final volume of 25 μl in detection antibody diluent.
Table 8. Preparing 1x detection antibodies from 20x stock (includes 25% excess volume).
5. After incubating the beads, samples, standards, blank, and controls,
slowly remove and discard the sealing tape.
6. Wash the plate three times with 100 µl wash buffer according to the
wash method of choice.
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 with a new sheet of sealing tape and incubate in the dark for
30 min at room temperature with shaking at 850 ± 50 rpm.
Prepare and Add Streptavidin-PE (SA-PE)
1. While detection antibodies are incubating, use Table 9 or the Calculation
Worksheet on page 34 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.
20x Detection Detection Antibody
# of Wells Antibodies, µl Diluent, µl Total Volume, µl
96 150 2,850 3,000
48 75 1,425 1,500