Bio-Rad Bio-Plex Assay Builder User Manual

Bio-Rad Laboratories, Inc.

2000 Alfred Nobel Dr.
Hercules, CA 94547 USA
1-800-424-6723 (in the US)

Bio-Plex Phospho-Histone H3

TM

Bio Plex

-

Phoshoprotein Detection

Instruction Manual

Now Includes Protocols for

Lysate Preparation!

4110018 Rev C 0406

For technical support, call your local Bio-Rad office, or
in the US, call 1-800-4BIORAD (1-800-424-6723)

For research use only. Not for diagnostic procedures.

Table of Contents

Section 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Section 2 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Section 3 Required and Recommended Materials . . . . .3

Section 4 Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Section 5 Lysate Preparation . . . . . . . . . . . . . . . . . . . . . 6

Section 6 Assay Instructions . . . . . . . . . . . . . . . . . . . . . 9

Section 7 Data Acquisition . . . . . . . . . . . . . . . . . . . . . . 15

Section 8 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . 20

Section 9 Safety Considerations . . . . . . . . . . . . . . . . . .23

Section 10 References . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Section 1
Introduction

Bio-Plex phosphoprotein assays and Bio-Plex total target assays are
bead-based multiplex assays (Luminex xMAP technology) that detect the
phosphorylation of proteins in lysates derived from cell culture or tissue
samples. These 96-well plate-format assays allow profiling of the specific
phosphorylation state of up to 100 different proteins using as few as two
wells and as little as 25 µl of lysate per well. The Bio-Plex total target
assay reports the abundance of the target protein in one well, while the
Bio-Plex phosphoprotein assay reports the level of phosphorylation of that
protein in a separate well. These instructions apply to both assays. For a
current list of all Bio-Plex phosphoproteins and total target assays, visit
www.bio-rad.com/products/phosphoproteins.

Available as Singleplex or Premixed Multiplex Assays

Bio-Plex phosphoprotein and total target assays are available as
singleplex or premixed multiplex assays. Singleplex assays are designed
to be flexible. They can be used individually to test for a single
phosphoprotein at a time, or they can be combined to create a multiplex
assay to test for a specific set of phosphoproteins in a single sample.
Premixed multiplex assays are the more convenient format for repeat
testing of a specific set of phosphoproteins. Both the coupled beads and
the detection antibodies are premixed and quality tested at Bio-Rad.
These assays are only available through Bio-Rad’s online x-Plex assay
service (

For research use only. Not for diagnostic procedures.

www.bio-rad.com/bio-plex/x-plex/).

1

Section 2

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Principle

The principle of these 96-well plate-format, bead-based assays is similar
to a capture sandwich immunoassay. An antibody directed against the
desired target protein is covalently coupled to internally dyed beads. The
coupled beads are allowed to react with a lysate sample containing target
protein. After a series of washes to remove unbound protein, a
biotinylated detection antibody specific for a different epitope is added to
the reaction. The result is the formation of a sandwich of antibodies
around the target protein. Streptavidin-phycoerythrin (streptavidin-PE) is
then added to bind to the biotinylated detection antibodies on the bead
surface.

Data from the reaction are then acquired using the Bio-Plex suspension
array system (or Luminex 100 system), a dual-laser, flow-based
microplate reader system. The contents of the well are drawn up into the
reader. The lasers and associated optics detect the internal fluorescence
of the individual dyed beads as well as the fluorescent signal on the bead
surface. This identifies each assay and reports the level of target protein
in the well. Intensity of fluorescence detected on the beads indicate the
relative quantity of targeted proteins. A high-speed digital processor
efficiently manages the data output, which is further analyzed and
presented as fluorescence intensity on Bio-Plex Manager
accompanying software package. If specific wells are identified for
comparison, the ratio of fluorescence intensity between those wells is
automatically calculated.

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Section 3
Required and Recommended
Materials

Required Materials

The following are required for phosphoprotein detection: 1) Bio-Plex
phosphoprotein or total target assays to test for specific target proteins in
lysate samples, 2) a Bio-Plex cell lysis kit to optimally lyse cell culture or
tissue samples, and 3) a Bio-Plex phosphoprotein detection reagent kit in
order to prepare the assays and acquire data on the instrument.

1

2

3

Bio-Plex phosphoprotein and total
target assays feature CST antibodies,
exclusively developed for Bio-Rad.

2

*Buffers contained in this kit have not been optimized for the use with
Bio-Plex cytokine assays.

3

Recommended Materials

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4

Section 4
Storage

Store the individual components as specified. Note that lysates are
shipped and stored separately from the coupled beads and detection
antibodies. Factors are shipped and stored separately from the cell wash
and cell lysis buffers.

5