MILLIPLEX MAP and Cell Signaling Pathways

Data SheetProduct Selection Guide

MILLIPLEX® MAP and Cell Signaling PathwaysYour Source for Luminex® xMAP® Technology

IRS-1

Src PI3K Ras

STAT5 STAT2

STAT6 FAK

FynJAK2

JAK1

ZAP70

LckLAT

STAT1

GSK3 Akt

mTOR

Rsk

Raf

Mek1/2

Erk1/2

STAT1 STAT3 Rsk CREB ATF1 Histone 3

Msk1

p70 56k

ProteinSynthesis

Transcription

Stress

β-Catenin

BAD

Ras MKK3 IKK

IκBα

PRAK

Hsp27

NFκB

p38p38 JNK Bcl2

cdc25c

cdc2

Aurora 2

PARP

ATF2 Jun Smad2/3

MKK3/6MKK4/7

Smad2/3

FRHD

MYPT1

Apoptosis

BAD

Caspase 8

Caspase 3

Syk

Death

FASLTNFαR

TGFβRCD79β

CD79α

TGFβ1,2,3

slg

CD3ζCD3ε

Cytokines TCR

Growth

IGF-RIns-R

c-Metc-Kit

PDGF-RαPDGF-RβEGF-R

Brown = Available from Millipore

www.millipore.com/milliplex_csp

http://www.millipore.com/?cid=BIOS-S-EPDF-1151-1107-DS

http://www.millipore.com/bmia/flx4/bmia_cellsignaling&cid=BIOS-S-EPDF-1151-1107-DS


MILLIPLEX MAP andCell Signaling PathwaysDetermining the complex interactions between cell signaling pathways is essential for understanding how the cell communicates with and responds to its microenvironment to maintain homeostasis.

Determining how the elements of signal transduction interact both

within and between pathways is essential to understanding normal

and disease processes. Using the Luminex xMAP platform, MILLIPLEX

MAP Cell Signaling offers you the ability to investigate simultaneously

the expression of intracellular total or phosphorylated proteins

involved in multi-pathway signaling or focused in a specific pathway.

You choose what best meets your needs: comprehensive panels in

a one-catalog-number, all-in-one kit format or an ever-expanding

MAPmate™ portfolio. No matter what you choose, you receive the

advantage of speed and sensitivity, as well as ease of use, beginning

with our simplified ordering process and including our user-friendly

protocols.

Available Cell Signaling Portfolio

•MILLIPLEXMAPCellSignalingKits

•MILLIPLEXMAPMAPmateProducts

•MILLIPLEXMAPBufferandDetectionKit

•CellLysates

•SpeciesCross-reactivity

•ComparisonData


AnexampleofthepowerofourMILLIPLEXMAPCellSignalingPortfoliocomparedtoWesternblotsis

demonstratedinthegraphsbelow.JurkatcellsweretreatedwithC305anti-TCRantibodyoranisotype-

matchedcontrolantibodyat2µg/mLovera10-minuteperiod.TheMILLIPLEXMAPHumanTCellReceptorKit

wasusedtomeasuretheeffectsoftheantibodyversuscontrolonthephosphorylationofpanelanalytesover

time.TheaverageMFIandstandarddeviationof4replicatewellsareshown.

Sincebandintensitiescorrelatewithfoldincreases,theWesternpresentedinthegraphsvalidatethe

multiplexdata.ThisfiguredemonstratestheadvantageofusingMILLIPLEXMAPCellSignalingMAPmatesand

kits:

•ResultsareeasiertoquantitatethanWesterns.

•Multipletargetscanbemeasuredsimultaneously.

The Effects of an Anti-TCR Antibody on Phosphorylation in Jurkat Cells using the Human T Cell Receptor Kit (Catalogue No. 48-690)

5432

00

1Ave

rage

MFI

(1000x)

Minutes of Stimulation

C305 Isotype Control

CD3ζ CD3ε

1 2 5 10

6789

10

2.52.01.51.0

00.5

3.03.54.04.55.0

10864

00

2Ave

rage

MFI

(1000x)



C305 anti-TCRβAntibody

Isotype ControlAntibody

1 2 5 10

1 minNT 2 min 5 min 10 min

1214161820

10

5

00

Ave

rage

MFI

(1000x)



LCK ZAP70



1 2 5 10


15

20

25

2.52.01.51.0

00

0.5Ave

rage

MFI

(1000x)





1 2 5 10


3.03.54.04.55.0

0

Ave

rage

MFI

(1000x)



LAT Erk



1 2 5 10


2.52.01.51.0

00.5

3.03.54.04.55.0

0

Ave

rage

MFI

(1000x)



CREB



1 2 5 10


5432

00

1Ave

rage

MFI

(1000x)





1 2 5 10


6789

10

5432

00

1Ave

rage

MFI

(1

00

0x)



CD3ζ CD3ε

1 2 5 10

6789

10

2.52.01.51.0

00.5

3.03.54.04.55.0

10864

00

2Ave

rage

MFI

(1

00

0x)





1 2 5 10


1214161820

10

5

00

Ave

rage

MFI

(1000x)



LCK ZAP70



1 2 5 10


15

20

25

2.52.01.51.0

00

0.5Ave

rage

MFI

(1000x)





1 2 5 10


3.03.54.04.55.0

0

Ave

rage

MFI

(1000x)



LAT Erk



1 2 5 10


2.52.01.51.0

00.5

3.03.54.04.55.0

0

Ave

rage

MFI

(1

000x)



CREB



1 2 5 10


5432

00

1Ave

rage

MFI

(1000x)





1 2 5 10


6789

10

5432

00

1Ave

rage

MFI

(1000x)



CD3ζ CD3ε

1 2 5 10

6789

10

2.52.01.51.0

00.5

3.03.54.04.55.0

10864

00

2Ave

rage

MFI

(1000x)





1 2 5 10


1214161820

10

5

00

Ave

rage

MFI

(1000x)



LCK ZAP70



1 2 5 10


15

20

25

2.52.01.51.0

00

0.5Ave

rage

MFI

(1000x)





1 2 5 10


3.03.54.04.55.0

0

Ave

rage

MFI

(1000x)



LAT Erk



1 2 5 10


2.52.01.51.0

00.5

3.03.54.04.55.0

0

Ave

rage

MFI

(1000x)



CREB



1 2 5 10


5432

00

1Ave

rage

MFI

(1000x)





1 2 5 10


6789

10

5432

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1Ave

rage

MFI

(1

00

0x)



CD3ζ CD3ε

1 2 5 10

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10

2.52.01.51.0

00.5

3.03.54.04.55.0

10864

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2Ave

rage

MFI

(1

00

0x)





1 2 5 10


1214161820

10

5

00

Ave

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MFI

(1000x)



LCK ZAP70



1 2 5 10


15

20

25

2.52.01.51.0

00

0.5Ave

rage

MFI

(1000x)





1 2 5 10


3.03.54.04.55.0

0

Ave

rage

MFI

(1000x)



LAT Erk



1 2 5 10


2.52.01.51.0

00.5

3.03.54.04.55.0

0

Ave

rage

MFI

(1000x)



CREB



1 2 5 10


5432

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1Ave

rage

MFI

(1000x)





1 2 5 10


6789

10

5432

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1Ave

rage

MFI

(1000x)



CD3ζ CD3ε

1 2 5 10

6789

10

2.52.01.51.0

00.5

3.03.54.04.55.0

10864

00

2Ave

rage

MFI

(1000x)





1 2 5 10


1214161820

10

5

00

Ave

rage

MFI

(1000x)



LCK ZAP70



1 2 5 10


15

20

25

2.52.01.51.0

00

0.5Ave

rage

MFI

(1000x)





1 2 5 10


3.03.54.04.55.0

0

Ave

rage

MFI

(1000x)



LAT Erk



1 2 5 10


2.52.01.51.0

00.5

3.03.54.04.55.0

0

Ave

rage

MFI

(1000x)



CREB



1 2 5 10


5432

00

1Ave

rage

MFI

(1000x)





1 2 5 10


6789

10

5432

00

1Ave

rage

MFI

(1

00

0x)



CD3ζ CD3ε

1 2 5 10

6789

10

2.52.01.51.0

00.5

3.03.54.04.55.0

10864

00

2Ave

rage

MFI

(1

00

0x)





1 2 5 10


1214161820

10

5

00

Ave

rage

MFI

(1000x)



LCK ZAP70



1 2 5 10


15

20

25

2.52.01.51.0

00

0.5Ave

rage

MFI

(1000x)





1 2 5 10


3.03.54.04.55.0

0

Ave

rage

MFI

(1000x)



LAT Erk



1 2 5 10


2.52.01.51.0

00.5

3.03.54.04.55.0

0

Ave

rage

MFI

(1000x)



CREB



1 2 5 10


5432

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1Ave

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MFI

(1000x)





1 2 5 10


6789

10

http://www.millipore.com/catalogue/item/48-690&cid=BIOS-S-EPDF-1151-1107-DS

Cell signaling, often called signal transduction, is a complex system of

communication that enables cells to perceive, communicate with and respond to

their microenvironment. Some communication requires direct cell-to-cell contact.

Other effects are induced when a receptor that has been activated by a ligand

interacts directly with other proteins inside the cell. Still other cellular changes are

the result of more complex signal transduction pathways—kinase receptors that,

when bound to a ligand, become phosphorylated. In this case, the phosphorylation

can generate a binding site for a different secondary protein, inducing a protein-

to-protein interaction that enables signal transduction. A terminal signaling event,

such as a transcription factor, is phosphorylated, resulting in the activation that

ultimately affects, for example, cell cycle progression. These multi-component cell

signaling pathways are so complex that often there is feedback, signal amplification,

and interactions between more than one signal and among several signaling

pathways.

The cell’s ability to respond to its environment is essential for normal

development, cellular repair and immunity and is the basis of homeostasis. Errors in

these pathways, caused by mutations that may result in the constitutive expression

or total inhibition of protein transcription, are responsible for such diseases as

diabetes, autoimmune disorders and cancer.

MILLIPLEX MAP Cell Signaling Kits and MAPmate Panels

AVAILABLE ANALYTES/THERAPEUTIC AREAS* Oncology Inflammation

Metabolic Syndrome Autoimmune Stem Cells Apoptosis CNS

Akt/PKB • • • • •

ATF2 • • • • • • •

BAD • • • •

Bcl-2 • • •

Caspase3 • • •

c-Jun • • • • • •

c-Kit • • • • •

c-Met/HGFR • • • • •

c-Met/HGFR • • • •

CREB • • • • • •

EGFR • • • •

Erb-B2/HER2 • • • •

ERK1/2 • • • • • • •

GSK3b • • • • •

HistoneH2A.X • • •

Hsp27 • • • • • •

IGF-1R • • • •

IkBa • • • • • • •

IR • • • • •

IRS1 • • • • •

JNK • • • • • • •

Lck • • • • •

MEK1 • • • • • • •

MKK6 • • • • • • •

MSK1 • • • • • • •

mTOR • • • • •

p38/SAPK • • • • •

p53 • • • •

p70S6Kinase • • • • •

PARP • • •

PDGFRa • • • • • •

PDGFRb • •

PRAS40 • • • • •

PTEN(Ser380) • • • • •

Rsk1 • • • •

STAT1 • • • • • •

STAT2 • • • • • •

STAT3 • • • • • •

STAT5A/B • • • • • •

STAT6 • • • • • •

ZAP-70 • • • •

*BasedoncitationfrequencyinPubMed.

MILLIPLEX MAP Cell Signaling Kits

AnalytesfeaturedinMillipore’sMILLIPLEXMAPCellSignalingKitshavebeenanalyticallyvalidatedtogether.Eachkitincludes

allthereagentsnecessarytorunanassayon3525-µLsamplesinduplicate—allinonekit,withonecataloguenumber:

• Beads(captureantibodies)

• Biotin(detectionantibodies)

• Lysisbuffer

• Assaybuffer(s)

• Stimulatedandunstimulatedcelllysates

(positiveandnegativecontrols)

• Streptavidin-Phycoerythrin

• Opaquemulti-screenplateandcover

• Mixingvials

• User-friendlyprotocol

• Environmentally-friendlypackaging

Multi-Pathway – 8-Plex (Catalogue No. 48-680)MILLIPLEXmapMulti-PathwayKitisan8-plexassaythat

examineseightphosphorylatedproteinsthatspan

severalsignaltransductionpathwaysincludingMAPK,

inflammationandapoptosis.

Analyte Phosphorylated

CREB (Ser133)

Erk/MAPK1/2 (Thr185/Tyr187)

IkBa (Ser32)

JNK (Thr183/Tyr185)


p38 (Thr180/Tyr182)

p70S6Kinase(Thr412)

STAT3 (Ser727)

STAT5A/B (Tyr694/699)

Human T Cell Receptor – 7-Plex (Catalogue No. 48-690)MILLIPLEXmapTCellReceptorKitisa7-plexassaythat

examinessevenphosphorylatedproteinsinvolvedin

immuneresponse,tumordevelopmentandinflammation.


CD3e (panTyr)

CREB (Ser133)

Erk/MAPK1/2 (Thr185/Tyr187)

LAT (panTyr)


Lck (panTyr)

Syk (panTyr)

ZAP-70 (panTyr)

Human Apoptosis – 3-Plex (Catalogue No. 48-670)Apoptosisplaysafundamentalroleinmanynormalbiologicalprocesses,includingdevelopment,homeostasisandDNArepair.

MILLIPLEXmapHumanApoptosisKitiscurrentlya3-plexassaythatexaminesactivecaspase-3andcleavedPARP,proteins

involvedinimmuneresponse,tumordevelopmentandinflammation.GAPDHisprovidedasacontrolforproteinloading.

AdditionalMAPmateproductsarealsoavailableforthestudyofapoptosis.

ActiveCaspase3 GAPDHActiveCleavedPARP

Analytes




Human MAP Kinase – 10-Plex (Catalogue No. 48-660)ThecomplexMAPKinasepathwayconnectsintracellular

responsestothebindingofgrowthfactorsviacell

surfacereceptors.TheMILLIPLEXmapMAPKinaseKitisa

10-plexassaythatexaminestenphosphorylatedproteins

involvedinnormalmitosisandtumordevelopment.


ATF2 (Thr69/71)

ERK/MAP (Thr185/Tyr187)Kinase1/2

HSP27 (Ser78)

JNK (Thr183/Tyr185)

cJun (Ser73)


MEK1 (Ser221)

Msk1 (Ser212)

p38 (Thr180/Tyr182)

p53 (Ser15)

STAT1 (Tyr707)

Human Src Family Kinase Family (SFK) – 8-Plex (Catalogue No. 48-650)TheSrcfamilyofproto-oncogenickinases(SFK)isthelargestnonreceptorproteintyrosinekinasefamilyconsistingof:

Src,Yes,Fyn,Fgr,Lck,Hck,Blk,andLyn.Expressedeitherubiquitouslyorinhematopoieticimmune-competentcells,

membersoftheSFKregulatesuchfunctionsasproliferation,adhesion,differentiation,andsurvivalthatplaycrucialroles

inembryonicdevelopmentandcellgrowth.Consequently,thesesignalingproteinsareoftenfundamentallyinvolvedinthe

progressionandoncogenesisoftumorssuchasnon-smallcelllungcancer(NSCLC),squamouscellheadandneckand

pancreaticcancers.

Human Akt/mTOR – 11-Plex (Catalogue No. 48-611)TheAkt/mTOR(mammaliantargetofrapamycin)pathway

playsacentralroleinanumberofcellularprocesses

includingtranslation,cellcycle,cellularenergyhomeostasis

andapoptosis.Keyserineandthreoninekinases,Aktand

mTORareactivatedviagrowthfactorsignaling,promoting

increasedproteintranslationandcellgrowth.Growthfactors,suchasinsulinandIGF,promotephosphorylationoftheIR,

IGF1RandIRS1,aswellasthedownstreamtargetsAkt,GSK3a,GSK3b,p70S6K,andRPS6.Constitutivephosphorylationis

observedwithPTEN,TSC2,andmTOR.ComparedtoWesternblotting,resultsfromtheAkt/mTORsignalingpanelweremore

quantitativeandsensitiveandsavedtimeandresourceswhenusedtoanalyzephosphorylatedproteinbiomarkersincellular

lysates.

Blk

Fgr

Lck

Lyn

Src

Yes

Fyn

Hck

Analytes (all pan Tyr)

2,000

p70S6

KIRS

GSK3α

IGF1R

GSK3β

AktPT

EN IRRPS

6TSC

2mTOR

0

MFI

4,000

6,000

HepG2 + InhibitorsHepG2 + Insulin

To demonstrate the power of the Akt/mTOR panel, HepG2 cells were treated with specific inhibitors such as wortmannin, which blocked phosphorylation of mTOR, TSC2, Akt, GSK3a, GSK3b, p70S6K and RPS6. This was followed by stimulation with 50 ng/mL IGF1 or 1 μM insulin for 15 minutes. Various phosphorylated biomarkers were simultaneously detected by the Luminex® xMAP® platform (represented as Average MFI) using Millipore’s Akt/mTOR panel.


Akt (Ser473)

GSK3a (Ser21)

GSK3b (Ser9)

IGF1R (Tyr1135/1136)

IR (Tyr1162/1163)

IRS1 (Ser312)


mTOR (Ser2448)

p70S6Kinase(Thr412)

PTEN (Ser380)

RPS6 (Ser235/236)

TCS2 (Ser939)




MILLIPLEX MAP MAPmate Portfolio: Building Custom Intracellular Assays

Ifyouprefertodesignacustomassay,Milliporehassimplifiedthisprocessforyou,makingorderingthenecessarykit

componentseasiersothatyouareassuredyouhaveeverythingyouneedtodoyourbestwork.

Plan your assay

Choosetheanalytesyouwanttoplextogetheranddeterminetheircompatibility.Keepinmindthatthe

followingcannotbeplexedtogether:

oTotalandphosphorylatedMAPmatepairs

oPanTyrandphospho-specificMAPmates

oPhospho-MAPmatesforasingletarget(Akt,STAT3,p53)

Usingthefollowingbuffertable,analytesthatusethesameassaybuffercanbeincludedinyourcustom

assay.Thisenablesyoutoconcentrateonproteinsbelongingtoaspecificsignaltransductionpathwayor

tolookatdownstreamtargetsthathavearoleinmultiplepathways.

STEP 1.

Order the MILLIPLEX MAP Cell Signaling Buffer and Detection Kit (Catalogue No. 48-602)

WiththeexceptionofMAPmates,thisbufferkitcontainseverythingyouneedtorunyourassay.

Thekitincludes:

• MILLIPLEXMAPLysisBuffer

• MILLIPLEXMAPAssayBuffer1

• MILLIPLEXMAPAssayBuffer2

• MILLIPLEXMAPAmplificationBuffer

• Streptavidin-Phycoerythrin

• Negative/positivecontrol(Phosphorylated/Total)

• Opaque96-wellFilterPlateandcover

• Emptymixingvials

(BufferanddetectionkitisrequiredforMAPmateanalysisofsamples.)

STEP 2.

Order the MAPmate products you have selected

AMAPmateconsistsof:

• CaptureAntibody(bead)

• DetectionAntibody(biotin)

• PositiveControl(stimulatedcelllysate)forphosphorylatedanalytes

Thepositivecontrolcanbeusedtoqualifyassayperformanceor,withserialdilutions,

todevelopastandardcurve.

STEP 3.

ORDERING IS AS EASY AS 1-2-3. TheMILLIPLEXMAPbufferkitandMAPmateproductswillprovideyouwithallthecomponentsand

reagentsyouneedtosimultaneouslydetectthemultipleanalytesyouhavechosentostudy.

http://www.millipore.com/bmia/flx4/bmia_cellsignaling#tab1=3&cid=BIOS-S-EPDF-1151-1107-DS

l Recommended • Acceptable 6 NotRecommended

Total MAPmate Portfolio

Catalogue No. Description Bead RegionLysis Buffer / Assay Buffer

1 2

46-605 TotalAkt/PKB,AssayBuffer1 38 l 6

46-675 TotalAkt/PKB,AssayBuffer2 62 6 l

46-657 TotalATF2 39 l l

46-695 TotalBad 59 l l

46-659 TotalBcl-2 61 l l

46-620 Totalc-Kit 8 l l

46-650 Totalc-Met/HGFR 46 l l

46-632 TotalCREB 37 l l

46-606 TotalEGFReceptor 34 l l

46-648 TotalErbB2/Her2 29 l l

46-609 TotalERK1/2 9 6 l

46-665 TotalHIF-1a 69 l l

46-608 TotalHsp27 50 • l

46-646 TotalIGF1R 48 l l

46-644 TotalIkBa 33 • l

46-687 TotalIR 65 l l

46-628 TotalIRS1 36 l l

46-618 TotalJNK 16 6 l

46-617 TotalLck 20 • l

46-669 TotalMEK1 45 l l

46-671 TotalMKK6 66 6 l

46-673 TotalMSK1 75 6 l

46-685 TotalmTOR 79 l l

46-621 Totalp21 28 l •46-612 Totalp38 54 6 l

46-662 Totalp53 53 6 l

46-630 Totalp70S6Kinase 19 l l

46-680 TotalPRAS40 55 l 6

46-678 TotalPTEN 63 • 6

46-654 TotalSTAT1 24 • l

46-634 TotalSTAT2 67 l l

46-625 TotalSTAT3 14 l •46-667 TotalHumanGAPDH 15 6 l

Forthemostupdatedversionofthistable,visitwww.millipore.com/milliplex_csp.Thenclickthe“MAPmateBufferTable”link.


































Phosphorylated MAPmate Portfolio

Catalogue No. Description Bead RegionLysis Buffer / Assay Buffer

1 2

46-604 ActiveCaspase3 6 l l

46-656 CleavedPARP(Asp214) 60 l l

46-601 PhosphoAkt/PKB(Ser473),AssayBuffer1 38 l 6

46-645 PhosphoAkt/PKB(Thr308),AssayBuffer1 38 l 6

46-677 PhosphoAkt/PKB(Ser473),AssayBuffer2 62 6 l

46-676 PhosphoAkt/PKB(Thr308),AssayBuffer2 62 6 l

46-658 PhosphoATF2(Thr69/71) 39 l l

46-694 PhosphoBad(Ser112) 59 l •

46-660 PhosphoBcl-2(Ser70) 31 l l

46-622 Phosphoc-Jun(Ser73) 52 l •

46-619 Phosphoc-Kit(Tyr) 8 l l

46-651 Phosphoc-Met/HGFR(Tyr) 46 l l

46-631 PhosphoCREB(Ser133) 37 l l

46-603 PhosphoEGFRec(Tyr) 34 l l

46-649 PhosphoErbB2/Her2(Tyr) 29 l •

46-602 PhosphoERK1/2(Thr185/Tyr187) 9 6 l

46-692 PhosphoHistoneH2A.X(Ser139) 43 l •

46-607 PhosphoHsp27(Ser78) 50 l l

46-647 PhosphoIGF1R(Tyr) 48 l l

46-688 PhosphoIR(Tyr1162/1163) 65 l l

46-643 PhosphoIkBa(Ser32) 33 • l

46-627 PhosphoIRS1(Tyr) 36 l l

46-613 PhosphoJNK/SAPK1(Thr183/Tyr185) 16 6 l

46-616 PhosphoLck(Tyr) 20 • l

46-670 PhosphoMEK1(Ser222) 45 • l

46-672 PhosphoMKK6(Ser207/Thr211) 66 • l

46-674 PhosphoMSK1(Ser212) 75 6 l

46-686 PhosphomTOR(SER2448) 79 l l

46-610 Phosphop38/SAPK(Thr180/Tyr182) 54 6 l

46-663 Phosphop53(Ser15) 53 • l

46-664 Phosphop53(Ser46) 53 • l

46-629 Phosphop70S6Kinase(Thr412) 19 6 l

46-614 PhosphoPDGFReceptora(Tyr) 21 • l

46-615 PhosphoPDGFReceptorb(Tyr) 32 • l

46-681 PhosphoPRAS40(Thr246) 55 l 6

46-679 PhosphoPTEN(Ser380) 63 • l

46-611 PhosphoRsk1(Ser380) 42 l •

46-655 PhosphoSTAT1(Tyr701) 24 • l

46-635 PhosphoSTAT2(Tyr690) 67 l l

46-624 PhosphoSTAT3(Ser727) 14 l •

46-623 PhosphoSTAT3(Tyr705) 14 l •

46-641 PhosphoSTAT5A/B(Tyr694/Tyr699) 35 • l

46-633 PhosphoSTAT6(Tyr641) 10 l l

46-661 PhosphoZAP-70(Tyr) 51 • l


l Recommended • Acceptable 6 NotRecommended














































MILLIPLEX MAP GAPDHUnique to Millipore

TheGlyceraldehyde3-PhosphateDehydrogenase(GAPDH)geneisconstitutivelyand

stablyexpressedinalmostalltissuesandcells,andisconsideredtobea

“housekeeping”gene.

MilliporehasdevelopedahumanGAPDHMAPmatetoaidyouinyourcellular

experiments,usingitasaloadingcontrolandforproteinnormalizationinyour

multiplexassays.

6,000

05 2.5

Concentration (µg/mL)

Ave

MFI

10 0.00

8,000

4,000

2,000

HeLa

HEK293

A431

A549

Kasumi

MCF7

GAPDH Human Lysate Titrationusing Assay Buffer 2 Total

GAPDH

Jurkat HeLa

Cell Lysates: Positive and Negative ControlsOurstimulatedandunstimulatedcelllysatesweredesignedtobeusedascontrols

withMILLIPLEXMAPCellSignalingKitsandMAPmateproducts.Theselysatesarealso

recommendedforuseinWesternblottingandimmunoprecipitation.

Available Lysates Available Lysates

Catalogue No. Control Sample Catalogue No. Control Sample

47-205 HeLa:unstim 47-216 MCF-7:IGF

47-206 Jurkat:unstim 47-217 Daudi:IL-4

47-207 Jurkat:anisomycin 47-218 A549:camptothecin

47-208 Jurkat:H2O

247-219 3T3:anisomycin

47-210 A431:EGF 47-221 3T3:unstim

47-211 HeLa:HS/Ars 47-222 HeLa:HGF

47-212 HeLa:TNFa 47-224 Ramos:Pervanadate

47-213 NIH3T3:PDGF 47-225 HL-60:Pervanadate

47-214 Kasumi-3:SCF 47-226 HeLa:IFNa

47-215 HEK293:LiCl

GAPDHcellularconcentrationscanvarysignificantlydependingonthecellline.Aninitialsampletitrationsimilartothatshownintherepresentativedataaboveisrecommendedforanypreviouslyuntestedcellline.




















3,000

00.03 0.06 0.13 0.25 0.50

µg of Cell Lysate per Well

MFI

0.01 0.02 1.00

4,0003,500

2,500

1,500

4,500

2,000

1,000500

Jurkat: unstimulated

Jurkat: anisomycin

MILLIPLEX MAP Detection of Cleaved PARP in Jurkat Cells

Western BlotCleaved PARP

– +anisomycin

IP/WesternCleaved PARP

– +anisomycin

ThegraphsbelowshowrepresentativedataobtainedwhenusingMillipore’scell

lysatesineachofthefollowingassays.Ineachcase,stimulatedandunstimulated

lysateswereresuspendedinultrapurewater.

• MILLIPLEX MAP: Aserialdilutionofarehydratedlysatewasanalyzedusingan

appropriateMAPmate.TheMedianFluorescenceIntensity(MFI)wasmeasuredwith

theLuminexInstrument.

• Western Blot: AliquotsoflysatewereanalyzedusingtheappropriateMAPmate

biotin-labeleddetectionantibodyandimagedusingstreptavidin-HRPand

chemiluminescence.

• Immunoprecipitation/Western: Aliquotsoflysatesweremixedwiththe

appropriateMAPmatecaptureantibodybeadstoimmunoprecipitatetheprotein.

TheimmunoprecipitatedproteinswereseparatedonSDS-PAGE,transferredto

nitrocellulose,andprobedwiththesameMAPmatebiotin-labeleddetection

antibody.Theproteinswereimagedusingstreptavidin-HRPandchemiluminescence.

3,000

00.03 0.06 0.13 0.25 0.50

µg of Cell Lysate per Well

MFI

0.01 0.02 1.00

4,0003,500

2,500

1,500

4,500

2,000

1,000500

Jurkat: unstimulated

Jurkat: anisomycin

MILLIPLEX MAP Detection of Cleaved PARP in Jurkat Cells

Western BlotCleaved PARP

– +anisomycin

IP/WesternCleaved PARP

– +anisomycin

Cell Lysates: Positive and Negative Controls (continued)

Total MAPmate Portfolio

Catalogue No. DescriptionSpecies Reactivity

Human Mouse Rat

46-605 TotalAkt/PKB,buffer1 l l l

46-675 TotalAkt/PKB,buffer2 l l l

46-657 TotalATF2 l l

46-695 TotalBad l l

46-659 TotalBcl-2 l

46-620 Totalc-Kit l

46-632 TotalCREB l l l

46-606 TotalEGFReceptor l

46-648 TotalErbB2/Her2 l

46-609 TotalErk1/2 l l l

46-665 TotalHIF-1a l l l

46-608 TotalHsp27 l

46-646 TotalIGF1R l

46-644 TotalIkBa l

46-628 TotalIRS1 l l l

46-618 TotalJNK l l

46-617 TotalLck l l

46-669 TotalMEK1 l l

46-650 Totalc-Met/HGFR l

46-671 TotalMKK6 l l l

46-673 TotalMSK1 l

46-621 Totalp21 l

46-612 Totalp38 l l l

46-662 Totalp53 l

46-630 Totalp70S6Kinase l l l

46-654 TotalSTAT1 l l

46-634 TotalSTAT2 l

46-625 TotalSTAT3 l l l

46-667 TotalHumanGAPDH l

46-687 TotalIR l l l

46-685 TotalmTOR l l l

46-678 TotalPTEN l l l

46-680 TotalPRAS40 l

Forthemostupdatedversionofthistable,visitwww.millipore.com/milliplex_csp.

Thenclickthe“SpeciesCross-ReactivityTable”link.

MILLIPLEX MAP Intracellular Assays: Species Cross-Reactivity

Whilemostofthecaptureanddetectionantibodiesareanti-human,manyare

cross-reactivewithotherspecies.Inmostcasestheseanalyteswereidentifiedby

examiningaminoacidsequencetodeterminehomology.Thetablebelowillustrates

speciescross-reactivityforeachMAPmateproduct.



































Phosphorylated MAPmate Portfolio

Catalogue No. DescriptionSpecies Reactivity

Human Mouse Rat

46-601 PhosphoAkt/PKB(Ser473),buffer1 l l l

46-677 PhosphoAkt/PKB(Ser473),buffer2 l l l

46-645 PhosphoAkt/PKB(Thr308),buffer1 l l l

46-676 PhosphoAkt/PKB(Thr308),buffer2 l l l

46-658 PhosphoATF2(Thr69/71) l l

46-694 PhosphoBad l l

46-660 PhosphoBcl-2(Ser70) l

46-604 ActiveCaspase3 l l

46-631 PhosphoCREB(Ser133) l l l

46-603 PhosphoEGFR(Tyr) l

46-649 PhosphoErbB2/Her2(Tyr) l

46-602 PhosphoErk1/2(Thr185/Tyr187) l l l

46-692 PhosphoHistoneH2A.X l l

46-607 PhosphoHsp27(Ser78) l

46-647 PhosphoIGF1R(Tyr) l

46-643 PhosphoIkBa(Ser32) l

46-688 PhosphoIR(Tyr1162/1163) l l

46-627 PhosphoIRS1(Tyr) l l l

46-613 PhosphoJNK/SAPK1(Thr183/Tyr185) l l

46-622 Phosphoc-Jun(Ser73) l l l

46-619 Phosphoc-Kit(Tyr) l

46-616 PhosphoLck(Tyr) l l l

46-670 PhosphoMEK1(Ser222) l l l

46-651 Phosphoc-Met/HGFR(Tyr) l

46-672 PhosphoMKK6(Ser207/Thr211) l l

46-674 PhosphoMSK1(Ser212) l

46-686 PhosphomTOR(Ser2448) l l l

46-610 Phosphop38/SAPK(Thr180/Tyr182) l l l

46-663 Phosphop53(Ser15) l

46-664 Phosphop53(Ser46) l

46-629 Phosphop70S6Kinase(Thr412) l l l

46-656 CleavedPARP(Asp214) l

46-614 PhosphoPDGFReceptora l l

46-615 PhosphoPDGFReceptorb l

46-681 PhosphoPRAS40(Thr246) l

46-679 PhosphoPTEN(Ser380) l l l

46-611 PhosphoRsk1(Ser380) l l l

46-655 PhosphoSTAT1(Tyr701) l l

46-635 PhosphoSTAT2(Tyr690) l

46-624 Phospho-STAT3(Ser727) l l l

46-623 Phospho-STAT3(Tyr705) l l l

46-641 Phospho-STAT5A(Tyr694) l l l

46-633 Phospho-STAT6(Tyr641) l

46-661 Phospho-ZAP70(Tyr) l















































MILLIPLEX MAP Comparative Data

PerformancecharacteristicsofMILLIPLEXMAPCellSignalingMAPmateproductswere

benchmarkedagainstanalytesselectedfromtwocompetitors.Atotaloffive

phosphoproteinanalytes(Table1)wereselectedbasedonavailabilityfromMillipore

andcompetitorcompanies,aswellasmaximalsignalingpathwayoverlap.

60

0

pEGF

R

pERK

1/2

p-p70

S6K

pJNK/S

APK1

p-p38

80

100

40

20

Millipore Competitor A Competitor B

60

0pERK1/2 p-p70 S6K pJNK/SAPK1 p-p38

80

100

40

20

150

0pEGFR

200

250

100

50


Comparison of Assaysusing Normalized Lysates

Comparison of Assays:Fold Induction

To determine fold induction the stimulated average MFI was divided by the unstimulated average MFI.

Protocol and Assay Design:oVerystraightforwardanduser-friendly

oNoadditionalsampletreatmentforsomeMAPmates

oOneprotocoldesignforallMAPmateproductsandkits

Kit Format: oAllkitsdesignedwiththesameformatformultiplexing

withtheleastoptimization

Buffers Required:oOnlytwobuffersrequired–DilutionandWashbuffers

Incubation Time and Temperature:

oOvernightincubationat4°C

oShorterincubationtimeatroomtemperaturepossible

Standards and Controls:oLyophilizednegativecontrol(unstimulatedlysate)

includedinMILLIPLEXMAPBufferandDetectionkit

oLyophilizedpositivecontrol(stimulatedlysate)included

withphospho-MAPmates

Advantages of MILLIPLEX MAP Cell Signaling Portfolio: Simpler, Easier Multiplexing

60

0

pEGF

R

pERK

1/2

p-p70

S6K

pJNK/S

APK1

p-p38

80

100

40

20


60

0pERK1/2 p-p70 S6K pJNK/SAPK1 p-p38

80

100

40

20

150

0pEGFR

200

250

100

50


Comparison of Assaysusing Normalized Lysates

Comparison of Assays:Fold Induction

To determine fold induction the stimulated average MFI was divided by the unstimulated average MFI.

Figure A and B.Manufacturer’sinstructionsforbothcelllysis,samplepreparationandforprotocolassayprocedureswerefollowedforeachofthecellsignalingkits(CatalogueNos.46-603,46-602,46-613,46-610,and46-629).Theresultsbelowshowingthefoldinductionvalues,aswellasfoldinductionwithnormalizedlysates,demonstratethesuperiorproductperformanceoftheMILLIPLEXMAPCellSignalingproductline.

Target Lysate Tested

pEGFR A431/EGF

pJNK/SAPK1 HeLa/HS-Ars

pERK1/2 A431/EGF

p-p38 HeLa/HS-Ars

p-p70S6K A431/EGF

Table 1.

A.

B.

xMAP TECHNOLOGY PROCESS FLOW

With xMAP technology, you will benefit from

the ability to simultaneously quantitate up to

100 analytes in a single sample, using as little

as 25 µL or less.

• Speed/High-Throughput: Simultaneously

measuretheconcentrationofalargenumberof

analytes,allowingyoutodomorefaster,gaining

earlyandcomprehensivedatasocriticaltoyour

work.

• Accuracy: xMAPtechnologygeneratesreal-time

analysisandaccuratequantificationofbiological

interactions.

• Reproducibility: Thehigh-volumeproduction

ofxMAPbeadsallowsassaystandardizationthat

solid-phasedflatarrayscannotprovide.

Luminex xMAP Technology

1 Luminexusesproprietarytechniquestointernallycolor-codemicrosphereswithtwofluorescentdyes.Throughpreciseconcentrationsofthesedyes,100distinctlycoloredbeadsetscanbecreated,eachofwhichiscoatedwithaspecificcaptureantibody.

3 Themicrospheresareallowedtopassrapidlythroughalaser,whichexcitestheinternaldyesmarkingthemicrosphereset.AsecondlaserexcitesPE,thefluorescentdyeonthereportermolecule.

2 Afterananalytefromatestsampleiscapturedbythebead,abiotinylateddetectionantibodyisintroduced.ThereactionmixtureisthenincubatedwithStreptavidinPEconjugate,thereportermolecule,tocompletethereactiononthesurfaceofeachmicrosphere.

Bead #12, IL-6Bead #29, IFN-γ

Bead #56, TNF-α

COMPARISON OF THE xMAP & WESTERN BLOT TECHNOLOGIESSide-by-sidecomparisonof

Multiplexingvs.WesternBlot

Traditional Method Multiplexing

“Western on a Bead”

Total Proteins

Phosphorylated Proteins

Western

1 Protein/probe

http://www.millipore.com/bmia/flx4/bmia_instruments&cid=BIOS-S-EPDF-1151-1107-DS

PRODUCTS • Higher Quality–Industry-leadingQC,QAandSixSigmaPrinciples.

• More Efficient–Analyticallyvalidatedkitsforconsistencyandreproducibility.

• All-in-One Kit–Asinglekitwithasinglecataloguenumber.

• More Flexible–Selectonlytheanalytesneededorchooseapre-mixedkit.

• Continuity of Knowledge Is the Key–Millipore“owns”everystepoftheprocess,

fromR&Dtomanufacturing.

• More Choices–ThebroadestrangeofmultiplexanalytesfortheLuminexxMAPplatform.

INSTRUMENTS • Speed/High-Throughput–Simultaneouslymeasuretheconcentrationofalargenumber

ofdifferentanalytes,allowingyoutodomorefaster,gainingearlyandcomprehensive

datasocriticaltoyourwork.

• Accuracy–xMAPtechnologygeneratesreal-timeanalysisandaccuratequantificationof

thebiologicalinteractions.

• Reproducibility–High-volumeproductionofxMAPmicrospheresallowsassay

standardizationthatsolid-phasedflatarrayscannotprovide.

• Low Sample Volume–Withminimalhands-ontime,youcanscreenmorethan

40analytesinasinglesampleusingaslittleas25µLorless.

SOFTWARE • TheLuminex xPONENT® softwarehelpsyoumanageandtrackallofyourmultiplex

assaysbycombiningeaseofuseandflexibilitytoperformbothproteinandnucleicacid

testingononesystem.It’salsocompatiblewiththeupcomingmagneticbead(MagPlex™

beads)andMILLIPLEXmagassays,andwillbemovingforwardwithcompatiblefuture

instrumentplatforms.

• NEW! MilliporenowoffersMILLIPLEX Analyst software,developedexclusivelyfor

MilliporebyVigeneTech®.Thisdataanalysispackagehelpsyoudoyourbestworkwith

increasedspeedandefficiency.MILLIPLEXAnalystsoftwareprovidesawholenewlevel

ofdataanalysisbyallowingyoutoimportlargedatasetsandbenefitfromtheabilityto

calculateyourresultsinseconds.Theuser-friendlyplatesetupfeatureletsyouwork

withspeedandefficiencywhiletheuniquealgorithmsoneventhemostchallengingdata

setsgiveyoutheperformanceyouneedformultiplexinganalysis.

SERVICES • Millipore’sBioMarkerServiceshasbeenperformingimmunoassaysforover25years.

• AssayservicesareavailableforRIAs,ELISAs,IRMAsandmultiplexassays,usingboth

Milliporekitsaswellasanycommerciallyavailableassays.

• Qualitycontrol,reproducibleresults,andconfidentialityareofparamountimportanceto

ourBioMarkerServicesteam.

• Millipore’sRegulatory Compliant Laboratoryisuniquelypositionedtoprovide

bioanalyticalservicestosupportyourpreclinicalandclinicalstudies.

MILLIPLEX MAP: Everything You Need—All In One Place

MILLIPLEX MAP

MILLIPLEX Analyzer

MILLIPLEX Analyst

BioPharma Services

Foracompleteandup-to-datelistofMILLIPLEXMAPanalytes,visitwww.millipore.com/analytes.

http://www.millipore.com/bmia/flx13/home&cid=BIOS-S-EPDF-1151-1107-DS

Description Catalogue No.

PhosphoAkt/PKB(Ser473),AssayBuffer1

46-601

PhosphoERK1/2(Thr185/Tyr187) 46-602

PhosphoEGFRec(Tyr) 46-603

ActiveCaspase3 46-604

TotalAkt/PKB,AssayBuffer1 46-605

TotalEGFReceptor 46-606

PhosphoHsp27(Ser78) 46-607

TotalHsp27 46-608

TotalERK1/2 46-609

Phosphop38/SAPK(Thr180/Tyr182)

46-610

PhosphoRsk1(Ser380) 46-611

Totalp38 46-612

PhosphoJNK/SAPK1(Thr183/Tyr185)

46-613

PhosphoPDGFReceptora(Tyr) 46-614

PhosphoPDGFReceptorb(Tyr) 46-615

PhosphoLck(Tyr) 46-616

TotalLck 46-617

TotalJNK 46-618

Phosphoc-Kit(Tyr) 46-619

Totalc-Kit 46-620

Totalp21 46-621

Phosphoc-Jun(Ser73) 46-622

PhosphoSTAT3(Tyr705) 46-623

PhosphoSTAT3(Ser727) 46-624

TotalSTAT3 46-625

PhosphoIRS1(Tyr) 46-627

TotalIRS1 46-628

Phosphop70S6Kinase(Thr412) 46-629

Totalp70S6Kinase 46-630

PhosphoCREB(Ser133) 46-631

TotalCREB 46-632


TotalSTAT2 46-634


PhosphoSTAT5A/B(Tyr694/Tyr699)

46-641

PhosphoIkBa(Ser32) 46-643

TotalIkBa 46-644

PhosphoAkt/PKB(Thr308),AssayBuffer1

46-645

Description Quantity Catalogue No.

Multi-Pathway8-Plex 96-wellplate 48-680

HumanTCellReceptor7-Plex 96-wellplate 48-690

HumanApoptosis3-Plex 96-wellplate 48-670

HumanMAPKinase9-Plex 96-wellplate 48-660

HumanSrcFamilyKinase-8-Plex 96-wellplate 48-650

HumanAkt/mTOR 96-wellplate 48-611

CellSignalingBufferandDetectionKit 96-wellplate 48-602


TotalIGF1R 46-646

PhosphoIGF1R(Tyr) 46-647

TotalErbB2/Her2 46-648

PhosphoErbB2/Her2(Tyr) 46-649

Totalc-Met/HGFR 46-650

Phosphoc-Met/HGFR(Tyr) 46-651

TotalSTAT1 46-654


CleavedPARP(Asp214) 46-656

TotalATF2 46-657

PhosphoATF2(Thr69/71) 46-658

TotalBcl-2 46-659

PhosphoBcl-2(Ser70) 46-660

PhosphoZAP-70(Tyr) 46-661

Totalp53 46-662

Phosphop53(Ser15) 46-663

Phosphop53(Ser46) 46-664

TotalHIF-1a 46-665

TotalHumanGAPDH 46-667

TotalMEK1 46-669

PhosphoMEK1(Ser222) 46-670

TotalMKK6 46-671

PhosphoMKK6(Ser207/Thr211) 46-672

TotalMSK1 46-673

PhosphoMSK1(Ser212) 46-674

TotalAkt/PKB,AssayBuffer2 46-675

PhosphoAkt/PKB(Thr308),AssayBuffer2

46-676

PhosphoAkt/PKB(Ser473),AssayBuffer2

46-677

TotalPTEN 46-678

PhosphoPTEN(Ser380) 46-679

TotalPRAS40 46-680

PhosphoPRAS40(Thr246) 46-681

TotalmTOR 46-685

PhosphomTOR(Ser2448) 46-686

TotalIR 46-687

PhosphoIR(Tyr1162/1163) 46-688

PhosphoHistoneH2A.X(Ser139) 46-692

PhosphoBad(Ser112 46-694

TotalBad 46-695

ORDERING INFORMATION

MILLIPLEX MAP MAPmate Products—96-well plate





















































































MILLIPLEX MAP Cell Lysates—96-well plateDescription Catalogue No.

HeLa:unstimulated 47-205

Jurkat:unstimulated 47-206

Jurkat:anisomycin 47-207

Jurkat:H2O2 47-208

A431:EGF 47-210

HeLa:HS/Ars 47-211

HeLa:TNFa 47-212

NIH3T3:PDGF 47-213

Kasumi-3:SCF 47-214


HEK293:LiCl 47-215

MCF-7:IGF 47-216

Daudi:IL-4 47-217

A549:camptothecin 47-218

3T3:anisomycin 47-219

Jurkat:paclitaxel 47-220

3T3:unstimulated 47-221

HeLa:HGF 47-222

Other Related Millipore ProductsoMILLIPLEXmapandMILLIPLEXmagCytokine/ChemokineMultiplex

Panels:human,highsensitivityhuman,non-humanprimate,

mouse,rat,canine

oMILLIPLEXmapEpiQuantPanels:human

oMILLIPLEXAnalyst

oBioMarkerServices

oKinaseProfiler™ServicesandProducts

oGPCRProfiler®ServicesandProducts

oCellSignalingAntibodies

Millipore,AdvancingLifeScienceTogether,MILLIPLEX,andGPCRProfilerareregisteredtrademarksofMilliporeCorporation.EpiQuant,KinaseProfiler,PathwayProfiler,MAPmate,andtheMmarkaretrademarksofMilliporeCorporation.Luminex,xPONENT,andxMAPareregisteredtrademarksofLuminexCorporation.Luminex200andMagPlexisatrademarkofLuminexCorporation.VigeneTechisaregisteredtrademarkofVigeneTech,Inc.Lit.No.PB2723EN00,Rev.BPrintedinU.S.A.andFrance07/10LS-SBU-09-02583©2010MilliporeCorporation,Billerica,MA01821U.S.A.Allrightsreserved.

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MILLIPLEX MAP and Cell Signaling Pathways

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