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.

TM

Software, the

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

Section 5

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Lysate Preparation

3. Prepare an adequate volume of lysing solution (refer to the table on
the left). For 10 ml of lysing solution, add 40 µl of
20 µl of
and set aside on ice. Then add 40 µl of 500 mM PMSF.

factor 2

to 9.9 ml of

cell lysis buffer

factor 1

. Vortex gently to mix

and

This section provides instructions for preparing lysates derived from cell
culture and tissure samples. For optimal recovery and sensitivity with the
phospho-Histone H3 assay, refer to suggested protocol for lysate
preparation.

1. Rinse the samples with

Adherent Cells — Stop the treatment reaction by aspirating the
culture medium and quickly rinsing the cells with ice-cold cell
wash buffer. The volume of cell wash buffer required is the
same as the volume of aspirated cell culture medium. Keep the
cells on ice.

Suspension Cells — Stop the treatment reaction by adding ice­cold wash buffer to the cells. The volume of cell wash buffer
required is twice that of the culture medium. Centrifuge the cells at
1,000 rpm for 5 min at 4ºC. Aspirate the supernatant.

Tissue Samples — Rinse the tissue sample with cell wash buffer
once. Cut the tissue into 3 x 3 mm pieces and transfer them to a
2 ml tissue grinder.

2. Prepare 500 mM PMSF by dissolving 0.436 g PMSF in 5 ml DMSO.
Store as 0.5 ml aliquots at –20ºC. Aliquots can be frozen and
thawed up to 5 times.

Lysing Solution Volume Guide

cell wash buffer

as follows:

4. Lyse the samples:

Adherent and Suspension Cells

a) Immediately add the lysing solution to the cells. The amount of

lysing solution needed depends on the cell concentration in the
culture vessel (see table on the left).

b) Agitate the cells as follows:

Culture Plate — For suspension cells, place the plate on ice and
pipet the contents of the wells up and down 5 times. For adherent
cells, scrape the cells with a cell scraper. For both, agitate the
plate on a microplate shaker at 300 rpm for 20 min at 4ºC.

Other Culture Vessel — Transfer the cell lysate to a centrifuge
tube and rotate for 20 min at 4ºC.

HINT: Freeze-thawing the lysate once using dry ice or a –20ºC
freezer may increase the extent of the lysis. Alternatively, briefly
sonicate (eg., with a Sonifier 450 as follows: Duty cycle = 40,
Output = 1, Pulse sonicating = 18 times).

c) Centrifuge the samples at 4,500 g for 20 min at 4ºC.

Tissue Samples

a) Immediately add 500 µl of lysing solution to the tissue grinder

and grind the tissue sample on ice using about 20 strokes.

b) Transfer the ground tissue to a clean microcentrifuge tube

and freeze the sample at –70ºC.

c) Thaw the samples, then sonicate on ice as suggested above.

6

d) Centrifuge the samples at 4,500 g for 4 min.

5. Collect the supernatant without disturbing the pellet.

6. Determine the lysate protein concentration. The protein
concentration should be 200–900 µg/ml. It may be necessary to
test-lyse your samples with different volumes of lysing solution to
obtain the specified protein concentration range.

7

7. Add an equal volume of

8. If the lysate is not tested immediately, store at –20ºC. The lysate is
stable for up to 5 freeze-thaw cycles.

Suggested protocol for lysate preparation of Histone H3 assay:

1. Follow steps 1–3 above

4. Lyse the samples:

a) Immediately add the lysing solution to the cells. The amount of

lysing solution needed depends on the cell concentration in the
culture vessel (see table on the left).

b) Briefly sonicate (e.g., with a Sonifer 450 as follows:

Duty cycle = 40, Output = 1, Pulse sonicating = two 10 min
pulses with a 1 min break in between).

c) Agitate the cells. Transfer the cell lysate to a centrifuge tube and

rotate for 20 min at 4

d) Centrifuge the samples at 4,500 g for 20 min at 4ºC.

5. Collect the supernatant without disturbing the pellet.

6. Determine the lysate protein concentration. The protein concentration
should be 200–900 µg/ml. It may be necessary to test-lyse your
samples with different volumes of lysing solution to obtain the
specified protein concentration range.

7. Add equal volume of assay buffer to the lysate.

8. Freeze (overnight) at -20ºC and thaw before testing.

assay buffer

.

ºC.

to the lysate.

Section 6
Assay Instructions

The following instructions apply to Bio-Plex phosphoprotein and total
target singleplex, custom-premixed, and mutliplex assays. Do not mix
phosphoprotein assays with its corresponding total target assays (e.g.
phospho-Akt and total Akt).

Plan Experiment

1. Assign which wells of a 96-well plate will be used for each lysate
(see the example below). Keep in mind that the instrument reads
wells down the plate and not across. Consider assigning the wells
vertically. A pullout worksheet has been provided in this manual that
may be used as a reference during the different assay steps.

Example Plate

2. Determine the total number of wells that will be used in the assay.
Include a 25% excess (or add 2 wells for every 8 wells used) to
ensure that enough diluted coupled beads, detection antibodies,
and streptavidin-PE are prepared. Record these numbers on the
worksheet since they will be referenced throughout the assay.

8

9

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Thaw Lysates

1. Retrieve the experiment lysates and lysates shipped with the assays
from –20ºC storage. These lysates were prepared using the protocol
in section 5 and contain 50% assay buffer.

Prepare Coupled Beads

Protect the beads from light by covering the tubes with aluminum foil.
Keep all tubes on ice until ready to use. Coupled beads must be mixed
manually prior to use when combining singleplex or premixed assays.

NOTE: Refer to the table provided with the lysate packaging to
identify which lysates shipped with the assays (visit www
rad.com/products/phosphoproteins/ to download the PDF). Select
the treated and untreated lysates from the table, which is used to
determine the assay performance of each Bio-Plex phosphoprotein
and total target assay. These should not be considered as
references. Instead, activation signals and ratios should be based on
experimental control lysates. For determining total protein

DC

concentrations, consider Bio-Rad’s

protein assay kit (Bio-Rad

catalog #500-0112).

2. Thaw the lysates at room temperature and then place them on ice.

3. If necessary, it is possible to further dilute the lysates. Lysing solution
freshly prepared (as specified in section 5) and assay buffer are
required. Use a 1:1 mixture of lysing solution and

assay buffer

further dilute the lysate.

10

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1. Vortex the coupled beads (50x) at medium speed for 5 sec.

2. Prepare a sufficient volume of coupled beads (1x) using

wash buffer

.
When preparing a multiplex assay, use equal volumes of each bead
(see sample below). Each well requires 1 µl of coupled beads (50x) for
each target adjusted to a final volume of 50 µl (refer to the table
below). These calculations can be done on the worksheet.

Volume of Coupled Beads (50x) in Each Well

to

Example Coupled Bead Calculations

11

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Calibrate Vacuum Apparatus

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The vacuum apparatus must be calibrated at the beginning of the assay
to ensure an optimal bead yield. For more detailed instructions, refer to
the Bio-Plex suspension array system hardware instruction manual.

1. Prewet all the wells of a 96-well filter plate with 100 µl of wash buffer.

2. Place the filter plate on the vacuum apparatus and turn on the
vacuum to the maximum level.

3. Press on the filter plate and note the time required to remove the
buffer from the wells by vacuum filtration. The evacuation time
should be 2–5 sec.

If the evacuation time is <2 sec, the pressure is too high. Open the
vacuum control valve slightly and repeat steps 1–3.

If the evacuation time is >5 sec, the pressure is too low. Close the
vacuum control valve slightly and repeat steps 1–3.

Assay Key

The following terms are repeated throughout the assay procedure. Refer
to these detailed instructions when

wash, rinse, incubate, and

vacuum-filter are shown in bold.

Assay Procedure

Bring all buffers to room temperature. Avoid bubbles when pipetting.

Wash the desired number of wells in a 96-well filter plate. If fewer

1.
than 96 wells are required, cover the unused wells with sealing tape
for later use.

2. Vortex the coupled beads (1x) for 5 sec at medium speed. Add
50 µl to each well and immediately

3.

Wash twice.

vacuum-filter.

4. Vortex the thawed lysates gently for 3 sec. Add 50 µl of lysate to
each well, changing the pipet tip after every volume transfer.

Incubate for 15–18 hr (or overnight).

Volume of Detection Antibodies (25x) in Each Well

Example Detection Antibody Calculations

12

13

5. The next day, prepare a sufficient volume of detection antibodies (1x)

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page). Each well requires 1µl of detection antibodies (25x) for each
target adjusted to a final volume of 25 µl (refer to the table on the
previous page).

6. After the incubation, slowly remove and discard the sealing tape,

7.

8. Vortex the detection antibodies gently and add 25 µl to each well,

9. After the incubation, slowly remove and discard the sealing tape,

10.

11. Keep the plate in the dark and prepare a sufficient volume of

Example Streptavidin-PE Calculations

vacuum-filter.

then

Wash 3 times.

changing the pipet tip after every volume transfer.

then

vacuum-filter.

Wash 3 times.

streptavidin-PE (1x) using
well requires 0.5 µl of streptavidin-PE (100x) adjusted to a final
volume of 50 µl. Store in the dark after preparation.

(see the example on the previous

Incubate for 30 min.

wash buffer

(see example below). Each

Section 7
Data Acquisition

Recommendations for acquiring data using the Bio-Plex suspension array
system are listed below. Alternatively, refer to the Bio-Plex Manager
software user guide or the instructions provided with the Luminex 100
instrument.

Prepare System

1. Empty the waste bottle and fill the sheath fluid bottle before starting.
This will prevent fluidic system backup and potential data loss.

2. Turn on the reader and microplate platform (and HTF if present).
Allow the system to warm up for 30 min.

3. Select Startup and follow the instructions to prepare the reader
to acquire data. If the system is idle for 4 hr the lasers will
automatically turn off and a 30 min warm-up period will again be
required prior to acquiring data. Select Warm up and wait for
the optics to reach operational temperature.

Calibrate With High RP1 Target Value

Calibrate using Bio-Plex calibration beads and target values. Daily
calibration is recommended before acquiring data.

1. Select Calibrate and confirm that the default values for CAL1
and CAL2 are the same as the values on the Bio-Plex calibration
bead labels. Use the Bio-Plex High RP1 target value for CAL2
calibration for Bio-Plex phosphoprotein and total target assays.

12. Vortex the diluted streptavidin-PE vigorously and add 50 µl to each

13. After the incubation, slowly remove and discard the sealing tape,

14.

15. Add 125 µl of

14

Incubate for 10 min.

well.

then

vacuum-filter.

Rinse 3 times.

If the data is not acquired immediately, the assay may be stored in
the dark at 4ºC for up to 24 hrs.

resuspension buffer

to each well. Incubate for 30 sec.

NOTE: When acquiring data for Bio-Plex phosphoprotein or total
target assays with a Luminex 100, Luminex Data Collector software,
and Luminex calibration beads, it is necessary to convert the Luminex
CAL2 calibration bead RP1 target value using the following equation:

Bio-Plex High RP1 target value = (Luminex RP1 target value) x 4.55

Add the new target value to the Luminex software by selecting
Calibrate, then New under the Reporter Channel in the Start
Calibration dialog. Enter the new target value and save it as a new
lot. Then calibrate using the new RP1 target value.

15

2. Select OK and follow the instructions for CAL1 and CAL 2
calibration.

Prepare Protocol

1. Open a new protocol by selecting File, then New from the main
menu. Locate the steps at the left of the protocol menu.

2. Select Step 1 (Describe Protocol) and enter information about the assay.

3. Select Step 2 (Select Analytes) and select the panel for
Phosphoproteins or Total Targets. Choose the target proteins for all
the assays on the plate. If both phosphoprotein and total target
assays are run on the same plate, two separate protocols must be
entered.

4. Select Step 3 (Format Plate) and click on the Plate Formatting tab.
Click on and drag the cursor over all the wells that contain
lysates.

Plate Formatting Example

5. Then select the Plate Groupings tab to display the plate grouping
tools.

a) Select Group and drag the cursor across all the wells to

define each assay group (exclude the ones that contain lysates
provided with the assays).

b) The first well of the group is automatically assigned as the

Reference well and the remaining wells in the group are Member
wells. To change the Reference well, select Reference and
click on the new Reference well.

c) Select Member/Reference or Reference/Member from the Ratio

pull-down list. When selecting the more common Member/Reference
option, the ratio of fluorescence intensity of each well will be
calculated against the fluorescence intensity of the Reference well.
This value will be reported as a ratio in the results file.

Plate Grouping Example

16

6. Select Step 6 (Enter Sample Info) and enter sample information. This
is the location where the wells are identified as containing either
Bio-Plex phosphoprotein or total target assays (see the example on
the right).

17

Sample Information Example

Acquire Data

1. Shake the assay plate at 1,100 rpm for 30 sec immediately before
acquiring data. Failure to do so will result in increased data
acquisition time due to bead settling.

2. Check that the filter plate is flat. While pressing on one end of the
plate, observe the distance that the opposite end of the plate is
raised off a flat surface. If the distance is >1 mm, transfer all contents
to a flat-bottom 96-well plate or another filter plate.

3. Visually inspect the plate and ensure that the assay wells are filled with
buffer prior to placing the plate in the Bio-Plex microplate platform.

6. If acquiring data from more than one plate, empty the waste bottle
and refill the sheath bottle after each plate. Select Wash Between
Plates and follow the instructions for fluidics maintenance. Then
repeat the Prepare Protocol and Acquire Data steps.

7. When data acquisition is complete, select Shut Down and
follow the instructions.

Reacquire Data

It is possible to acquire data from a well or plate a second time using the
Rerun/Recovery mode located below Start in Step 7 (Run Protocol).

1. Check the wells where data will be acquired a second time.
Any previous data will be overwritten.

2. Remove the buffer by vacuum filtration and add 125 µl of
resuspension buffer to each well. Cover the filter plate with a new
sheet of sealing tape.

3. Repeat

Acquire Data steps 1–6 to acquire data a second time.

The data acquired should be similar to the data acquired initially;
however, the data acquisition time will be extended since fewer
beads are present in each well.

4. Slowly remove the sealing tape and any plate cover before placing
the plate in the reader.

5. Select Step 7 (Run Protocol):

a) Specify data acquisition for

25 beads per region.

b) In Advanced Settings, confirm that the default DD gate values

are set to 4335 (low) and 10000 (high).

NOTE: When using a Luminex 100 instrument, set the gates
according to the Luminex procedure located in the manual.

c) Select Start and save the .rbx file. Then follow the instructions for

data acquisition.

18

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Section 8
Troubleshooting

This troubleshooting section addresses problems that may be encountered
with Bio-Plex phosphoprotein or total target assays. If the problems listed
below are encountered, review the possible causes and solutions provided.
This will assist in resolving problems directly related to the assay. Use the
Bio-Plex validation kit to validate all the key functions of the array reader
and assist in determining whether or not the array reader is functioning
properly.

Possible Causes Possible Solutions

Filter Plate Leakage

Vacuum setting too high

Low Signal (Good Signal From
Lysates Provided with the Assays but
Weak or No Signal From Experiment
Lysates)

Protein concentration in
lysate too low or too high

Low Signal (Weak or No Signal From
Lysates Provided with the Assays and
Experiment Lysates)

Detection antibody and/or
streptavidin-PE diluted incorrectly

This could result in tearing of the filter.
Confirm that the vacuum pressure is
set as specified in the vacuum
calibration procedure section. Also
refer to the Vacuum Manifold Setup
in the Bio-Plex suspension array
system hardware instruction manual.
Use the recommended filter plate
vacuum apparatus

Verify the protein concentration in the
cell lysate samples. Adjust the
amount of lysing solution used in the
lysate preparation to achieve an
optimal protein concentration of
200–900 µg/ml prior to adding an
equal part of assay buffer

Check the calculations and be careful
to add the correct volumes for dilution

Possible Causes Possible Solutions

Expired beads, detection antibody,

Use new or unexpired components

and/or streptavidin-PE used

Incorrect incubation temperature
used during incubation steps

Incubation time insufficient

Incubations should be at room
temperature (20–22ºC)

Adhere to the recommended
incubation times

Low Bead Count

Cell debris in lysate not
cleared

Remove the cellular debris by
centrifugation at 4,500 g for 20 min at
4ºC. Avoid disturbing the pellet while
collecting the supernatant

Resuspension buffer not used
after streptavidin-PE incubation

Rinse the beads 3 times with
resuspension buffer after the
streptavidin-PE incubation step

Vacuum setting too high

This results in bead loss. Calibrate the
vacuum apparatus as specified

Filter plate not shaken enough
before each incubation step
and prior to data acquisition

Reader clogged

Shake the filter plate at 1,100 rpm for
30 sec before each incubation and
immediately before acquiring data

Refer to the troubleshooting guide in the
Bio-Plex hardware instruction manual

High Coefficient of Variation (CV)

Plate sealer reused

This could result in contamination. Use
a new sheet of sealing tape for each
incubation

Buffer not completely filtered
from wells

Be sure that the wells are filtered
completely and that no residual volume
remains

20

21

Possible Causes Possible Solutions

Contamination with wash buffer
during wash steps

Microplate shaker set to an
incorrect speed

Cellular debris not cleared from
lysate

During the wash steps, do not splash
wash buffer from one well to another.
Filter the wells completely so that no
residual volume remains. Also, be sure
that the microplate shaker setting is not
too high. Reduce the microplate shaker
speed to minimize splashing

Check the microplate shaker speed
and use the recommended setting.
Setting the speed too high may cause
splashing and contamination. Use the
recommended plate shaker

Be sure to remove cellular debris by
centrifugation as directed. Avoid
disturbing the pellet while collecting the
supernatant

Section 9
Safety Considerations

Eye protection and gloves are recommended while using this product.
Consult the MSDS for additional information.

Human Source Material. Treat As Potentially Infectious.

The lysates provided with Bio-Plex phosphoprotein and total target assay
contain components of human origin. The components are known to
contain an agent that requires handling at Biosafety Level 2 containment
[US Government Publication: Biosafety in Microbiological and Biomedical
Laboratories (CDC, 1999)]. These agents have been associated with
human disease. These components have not been screened for hepatitis
B, human immunodeficiency viruses, or other adventitious agents.
Handle Bio-Plex phosphoprotein positive and negative controls as
potentially biohazardous material under at least Biosafety Level 2
containment.

Bead resuspension buffer not used
after streptavidin-PE incubation

High Background Signal
(From Both Lysates Provided with
the Assays and Experiment Lysates)

Vacuum pressure too low,
resulting in residue in wells

Wash steps performed incorrectly

Streptavidin-PE incubation
step too long

22

Wash 3 times with bead resuspension
buffer after streptavidin-PE incubation
as described in the assay instructions

Use recommended filter plate vacuum
apparatus with proper vacuum pressure
setting

Perform washes as described in the
assay instructions

Check suggested incubation times for
appropriate steps of the assay. Follow
the suggested time for incubation

23

Section 10
References

Fulton R, McDade R, Smith P, Kienker L, and Kettman J Jr, Advanced
multiplexed analysis with the FlowMetrix system, Clin Chem 43,
1749–1756 (1997)

Chang L, and Karin M, Mammalian MAP kinase signalling cascades,
Nature 410, 37–40 (2001)

For a complete list of publications using Bio-Plex Phosphoprotein
Detection Assays, refer to bulletin 5394.

xMAP is a trademark of Luminex Corp.

By purchasing this kit, which contains fluorescent labeled microsphere beads authorized by
Luminex, you, the customer, acquire the right under Luminex's patent rights* to use this kit or
any portion of this kit, including without limitation the microsphere beads contained herein, only
with Luminex’s laser-based fluorescent analytical test instrumentation known under the name of
Luminex 100, for example as marketed by Bio-Rad Laboratories, Inc. in the Bio-Plex system.

*Including, but not limited to US patent 5,981,180; 6,046,807; 6,057,107.

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