Bio-Rad Bio-Plex Pro Human Angiogenesis Reagent and Diluent Kits - Manual (Page 16)

14
Assay Procedure
Bring all buffers to room temperature. Avoid bubbles when pipetting.
Assay Key – The following terms are repeated throughout the assay
procedure. Refer to these detailed instructions when wash, incubate, and
vacuum-filter are shown in bold.
1. Equilibrate the diluted standards, samples, and controls at room
temperature for 20 min prior to use.
2. Prewet and block the desired number of wells in a 96-well filter plate
with 100 µl of assay buffer and vacuum-filter. If fewer than 96
wells are required, mark the plate to identify the unused wells for
later use and cover the unused wells with sealing tape.
3. Vortex the coupled magnetic beads (1x) for 15–20 sec at medium
speed. Add 50 µl to each well and immediately vacuum-filter.
4. Wash twice.
5. Gently vortex the diluted standards, controls, and samples for 1–3 sec.
Add 50 µl of standard, control, or sample to each well, changing the
pipet tip after every volume transfer.
Incubate for 30 min.
6. While the samples are incubating, perform a 30 sec quick-spin
centrifugation of the detection antibody (10x) prior to pipetting to
collect the entire volume at the bottom of the vial.
Add 100µL of wash buffer to each well. Place the filter plate on a calibrated vacuum
apparatus and remove the buffer by vacuum filtration. Blot the bottom of the filter plate
with a clean paper towel. Repeat as specified.
Gently cover the filter plate with a new sheet of sealing tape. Place the filter plate on a
microplate shaker and then cover with aluminum foil. Shake the filter plate at room
temperature at 1,100 rpm for 30 sec, then at 300 rpm for the specified incubation time.
Tip: Thoroughly blot the bottom of the filter plate with a clean paper towel to
prevent cross-contamination and plate leakage.
Tip: Apply sealing tape gently on the filter plate (e.g. press down only on edges) to
prevent positive pressure inside the wells that could lead to plate leaking during shaking.
To avoid splashing of samples that may lead to cross-well contamination, slowly ramp
up the shaker to the maximum speed before incubation. Cover plate with aluminum foil
to prevent photobleaching.
Place the filter plate on a calibrated vacuum apparatus and remove the buffer by
vacuum filtration. Blot the bottom of the filter plate with a clean paper towel.
Tip: Visually examine each well to ensure that buffers are completely drained from each well.