Bio-Plex Pro™ RBM Apoptosis Assays
Quick Guide
For Use With Instruction Manual #
Bio-Plex Pro
This guide can be used to prepare and run a full 1 x 96-well assay plate.
For more information on a given step, refer to the corresponding section
of the complete instruction manual. New users can download the manual,
which includes detailed instructions and a list of kit components, at
www.bio-rad.com/bio-plex.
IMPORTANT! Pay close attention to vortexing, shaking, and incubation instructions.
Deviation from the protocol may result in low assay signal and assay variability.
A. Reagent Preparation
1. Reconstitute the following lyophilized reagents in dH20 before use
™
RBM Apoptosis As says 100 33631
according to the table below.
Reagent Volume, µl Reagent Volume, ml
Standards mix 150 Blocking buffer 1.5
Control 1 100 Standard diluent 1.0
Control 2 100 Detection antibodies 4.8
a. Allow vial to sit at room temperature for a minimum of 5 min, not to
exceed 30 min.
b. Mix by vortexing at a medium setting.
2. Bring the 10x assay buffer to ambient temperature (RT).
a. Mix by inversion to ensure all salts are into solution.
b. Prepare 1x assay buffer — dilute 1 part 10x assay buffer (60 ml) with
9 parts of dH
0 (540 ml).
2
Bio-Plex Pro Assay Quick Guide
B. Dilution of Standard (1:3 Serial Dilution)
1. Label 8 polypropylene tubes S1 through S8.
2. Transfer the reconstituted standard into the tube labeled “S1.”
3. Add the appropriate amount of the standard diluent into the labeled
tubes according to the table below (this will be sufficient for duplicate
standard curves and blanks).
Standard Volume of Standard Diluent, µl Volume of Standard, µl
S2 100 50 of S1
S3 100 50 of S2
S4 100 50 of S3
S5 100 50 of S4
S6 100 50 of S5
S7 100 50 of S6
S8 100 50 of S7
Blank 100 —
4. Prepare working standards (S2–S8) by serial dilution. Transfer the
appropriate volume of standard into each of the labeled tubes with
standard diluent as outlined above.
5. Vortex each standard at a medium setting before proceeding with the next
serial dilution. Change pipet tip at each dilution step.
C. Sample Preparation
1. Refer to instruction manual #10033631 for detailed sample preparation.
Note: Pay close attention to cell lysis, homogenization, and fractionation
protocols in the manual. Analytes of interest may be localized to the
cytosolic fraction or to the nuclear and mitochondrial fraction.
2. Prepare sample dilutions in 0.5 ml or 1.0 ml polypropylene tubes as
required for the assay.
Bio-Plex Pro Assay Quick Guide
3. For sample analysis, bring samples to a final protein concentration of
500 µg/ml by diluting with lysis dilution buffer (LDB). Further dilution may
be necessary for targets with high expression levels.
4. Centrifuge samples at 500 x g for 5 min to remove particulates prior to use.
Note: Controls are ready to use after reconstitution. No further dilution
is needed.
D. Dispensing of Reagents
1. Add 10 µl of blocker to all wells of the plate.
2. Add 30 µl of the standard, control, sample, or blank to the appropriate
well of the plate.
3. Vortex the capture beads at medium speed for 10–20 sec. Add 10 µl of
the beads to all wells of the plate.
4. Cover plate with plate seal and protect from light with aluminum foil.
Incubate on shaker at 850 ± 50 rpm for 1 hr at RT.
5. Wash the plate three times with 100 µl 1x assay buffer.
6. Vortex the reconstituted detection antibodies at medium speed for
10–20 sec. Add 40 µl to each well.
7. Cover and incubate at 850 ± 50 rpm, as in Step 4, for 1 hr at RT. Do not
aspirate after incubation.
8. Prepare the required dilution of streptavidin-PE (SA-PE) as outlined in the
following table.
Note: Volumes in the table are for an entire 96-well plate. Smaller volumes
can be prepared, provided that the dilution ratios are maintained.
9. Add 20 µl of diluted SA-PE to the required plate wells.
Volume of Volume of
SA-PE Dilution SA-PE, µl 1x Assay Buffer, µl Total Volume, µl
1:10 225 2,025 2,250
Bio-Plex Pro Assay Quick Guide
10. Cover and incubate at 850 ± 50 rpm, as in Step 4, for 30 min at RT.
11. Wash the plate three times with 100 µl 1x assay buffer.
12. After the final wash, resuspend the beads in 100 µl 1x assay buffer. Cover
plate as in Step 4 and shake the plate at 850 ± 50 rpm for 30 sec.
®
13. Remove the plate seal and read plate at low PMT (Bio-Plex
standard PMT (Bio-Plex 3D), or default settings (Bio-Plex
The Bio-Plex suspension array system includes fluorescently labeled microspheres and
instrumentation licensed to Bio-Rad Laboratories, Inc. by Luminex Corporation.
Bio-Plex Pro RBM kits are manufactured by Myriad RBM.
200),
®
MAGPIX™).
P10033632
Life Science
Group
Bio-Rad
Laboratories, Inc.
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