Bio-Rad Bio-Plex Pro™ Human Th17 Cytokine Assays - Manual (Page 18)

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2. Determine the total protein concentration of the lysate. It may be
necessary to test lyse your samples with different volumes of lysing
solution to obtain the specified protein concentration range.
3. Dilute at least 1:2 in sample diluent + 0.5% BSA, to a final protein
concentration of 50–500 µg/ml.
For optimum antibody binding during sample incubation, it is
important to dilute lysates as much as possible to reduce the
detergent concentration.
4. If the lysate is not tested immediately, store at –20°C. Lysates are
stable for up to five freeze-thaw cycles.
6. Prepare Coupled Beads
1. Use Tables 6–7 or the Calculation Worksheet on page 35 to calculate
the volume of coupled beads and assay buffer needed. Tables 6–7
include ~20% excess to compensate for transfer loss.
2. Add the required volume of Bio-Plex ® assay buffer to a 15 ml
polypropylene tube.
3. Vortex the 20x stock of coupled beads at mid speed for 30 sec.
Carefully open the cap and pipet any liquid trapped in the cap back
into the tube. This is important to ensure maximum bead recovery.
Do not centrifuge the vial; doing so will cause the beads to pellet.
4. Dilute coupled beads to 1x by pipetting the required volume into the
15 ml tube. Vortex.
Each well of the assay requires 2.5 μl of the 20x stock adjusted to a
final volume of 50 μl in assay buffer.
5. Protect the beads from light with aluminum foil. Equilibrate to room
temperature prior to use.