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

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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 9. 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.
10. Wash the plate three times with 100 µl of wash buffer according
to the wash method of choice.
11. To resuspend beads for plate reading, add 125 µl assay buffer to
each well. Cover the plate with a new sheet of sealing tape. Shake
at room temperature at 850 ± 50 rpm for 30 sec and slowly remove
the sealing tape. Ensure that the plate cover has been removed
before placing the plate on the reader.
# of Wells 100x SA-PE, µl Assay Buffer, µl Total Volume, µl
96 60 5,940 6,000
48 30 2,970 3,000