Bio-Rad Human MMP and TIMP Assays - Manual (Page 22)

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Prepare and Add Detection Antibodies
1. While the samples are incubating use Tables 9 and 10 or the
Calculation Worksheet on pages 35–36 to calculate the volume
of detection antibodies and Bio-Plex detection antibody diluent
HB needed to prepare a 1x stock. Detection antibodies should be
prepared 10 min before use.
2. Add the required volume of Bio-Plex detection antibody diluent HB to
a 5 ml polypropylene tube.
3. Vortex the 10x stock of detection antibodies for 15–20 sec at
medium speed, then perform a 30 sec spin to collect the entire
volume at the bottom of the tube.
4. Dilute detection antibodies to 1x by pipetting the required volume into
the 5 ml tube. Vortex.
Each well of the assay requires 2.5 μl of the 10x stock adjusted to a
final volume of 25 μl in detection antibody diluent HB.
Preparing 1x detection antibodies from 10x stock (includes 25% excess volume)
Table 9. Premixed panel or one singleplex assay.
Table 10. Mixing singleplex assays.
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 per well.
Singleplex #1 Singleplex #2 Detection
10x Detection 10x Detection Antibody
# of Wells Antibodies, µl Antibodies, µl Diluent, µl Total Volume, µl
96 300 300 2,400 3,000
48 150 150 1,200 1,500
10x Detection Detection Antibody
# of Wells Antibodies, µl Diluent, µl Total Volume, µl
96 300 2,700 3,000
48 150 1,350 1,500