Bio-Rad Apoptosis Assays User Manual

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.