Signaling - Antibodies, Proteins, Kit and Assays Product Selection Guide

8/6/2019 Signaling - Antibodies, Proteins, Kit and Assays Product Selection Guide

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Activate Your Discovery Network.Antibodies, small molecule inhibitors, kits,assays and proteins or signaling research.

Product Selection Guide

EMD Millipore is a division o Merck KGaA, Darmstadt, Germany
http://www.millipore.com/?cid=BIOS-S-EPDF-1111-1208-RChttp://www.millipore.com/antibodies/flx4/cellsignaling?cid=BIOS-S-EPDF-1111-1208-RC


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CALBIOCHEMSMALL MOLECULES

Small-moleculecompounds, includinginhibitors, activators, andother pathway modulators,are critical tools orresearchers studyingcell signaling. Chemicalgenetics, in which losso unction is imposedusing small molecules, canreveal connections withinsignaling networks. EMDMillipores Calbiochemreagents have been citedin thousands o peer-reviewed publications.From libraries and pathwaypanels to individualreagents, the Calbiochemline o products o ersthe widest and most cited

selection o inhibitors andactivators worldwide.

Plat orms, Technologies,

and ServicesAs a tools provider and partner in research, EMD Millipore is committed to the advancement o li e science researchand therapeutic development. This guide includes a number o new products or target identi cation, pathwaydetection, and pro ling. These products provide proven solutions or a range o applications and are backed byextensive technical support.

ANTIBODIES ANDIMMUNOASSAYS

With the expertise o Upstate and Chemicon,EMD Millipore providesan extensive, ocused,validated port olioo antibodies andimmunoassays, withbreadth and depth inmajor research areasbacked by excellentservice and support. EMDMillipore also o ers avariety o ELISAs in majorresearch areas, includingcell signaling, and noveltools or improving theWestern blotting workfow.

CELL-BASEDASSAYS

Our port olio o live cell,whole-cell and cell-based activity assays andreporter systems advancesdirect and indirectdetection o cell signaling.These technologies

acilitate protein targetvalidation, identi y cellularpathways and determinemechanism o action

or lead optimizationenvironments.

FLOW CYTOMETRY ASSAYS AND

SYSTEMSSimultaneously measuringmultiple parameters onindividual cells, fowcytometry is essential orin-depth cell analysis.Our Amnis imaging fowcytometers combine thespeed, sensitivity, andphenotyping abilities o fow cytometry with theimagery and unctionalinsights o microscopy,taking cell signalingstudies to higher levelso discrimination anddiscovery. Our easyCytefow cytometers provideprecise measurement viamicrocapillary technologythat translates into smaller

samples, less reagents,and minimal waste. Validated FlowCellectassay kits, Milli-Markconjugated antibodiesand application-speci cso tware modules providea complete solution orfow cytometry.


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MILLIPLEXmap MULTIPLEX ASSAYS

MILLIPLEXmap assayso er the broadestselection o multiplexkits and reagents in awide variety o researchareas, measuring multiplebiomarkers using a smallsample size. MILLIPLEXmap enables thesimultaneous detectiono multiple soluble orintracellular biomarkers.Using the LuminexxMAP bead-basedtechnology, these fexibleand customizable assaysare exhaustively tested andquali ed or sensitivity,speci city, reproducibilityand wide dynamic range.Providing absolute,

site-speci c quantitationo phosphorylation, theMILLIPLEXmap EpiQuantassays are the mostadvanced plat orm or cellsignaling analysis.

MOLECULARBIOLOGY

AND PROTEINPREPARATIONFor every step o themolecular biologyand protein workfow,

rom cloning DNAtargets to puri ying andconcentrating recombinantproteins, EMD Milliporeprovides reagents, kits,cells and tools that arespeci cally designed tomeet your scienti c andtechnical goals. For proteinquantitation, the in rared-based Direct Detectspectrometer distinguishesproteins and peptides

rom inter ering samplecomponents, providingmore accurate results

without the pit alls o colorimetric assays.

CELLS AND CELLCULTURE

EMD Millipores innovativecell culture solutions helpoptimize cell growth andmaintenance or signalingresearch. We o er anextensive range o humanand rodent stem cells,primary cells and mediadesigned or most typeso stem cells, includingembryonic, mesenchymal,and neural stem cells. Ourfexible sterile ltrationdevices o er ast fowand have many membraneoptions. Also availableare microfuidic systemsand cultureware to mimicin vivo conditions andprovide coculture options.


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Introduction

Cell signaling, o ten called signal transduction, is a complex network o receptors,enzymes, and messengers that enables cells to perceive, communicate with andrespond to their environment. Cell signaling controls and regulates every aspect o cell unction, including cell division, cell proli eration, and cell death.

Cell signaling may occur in three orms: Extracellular signals, such as contact

with other cells, hormones, growth

actors, chemoattractants, metal ions,

or contact with extracellular matrix (ECM)

components. Interaction betweencytosolic signaling

proteins in response to either intracellular

molecules or messengers generated as a

result o ligand binding to transmembrane

receptors. Nuclear response to stimuli, ultimately

mani ested as changes in gene expression.

Despite the intricate and complex nature o

signaling networks, the tools used to study

them are based on the classic principles

o protein detection and manipulation.

Immunodetection has undergone

technological advances, such as multiplex

protein detection and multiparametric cell

analysis. Nevertheless, the success o cell

signaling research still largely depends on

the quality, sensitivity, and speci city o

the antibodies used or detection and small

molecules to induce or block modi cations inenzyme activities.

Combining its long-trusted immunodetection

tools and reagents with the expertise o

Calbiochem, Upstate, Chemicon and

Linco, EMD Millipore is the leading partner

or scientists in both basic cell signaling

research as well as signaling pathway pro ling

or the drug discovery industry. By using this

product guide in conjunction with our online

resource (www.millipore.com/signaling), let

us help you design an experimental strategy

or understanding the signaling networks in

your biological model o interest.
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Growth Factor SignalingGrowth actors are peptides that are secreted by a varietyo cells, act through cell sur ace receptors and can elicit

similar as well as distinct biological responses in theirtarget cells. Growth actors can regulate cell growth,proli eration, di erentiation and maturation, makinggrowth actor signaling ideal or targeting in research,discovery and clinical environments. Major amilies o

Anti-VEGF

(Catalogue No. ABS82) VEGF (Vascular endothelial growth actor, VEGFA, VEGF-A), a dimeric ligand, is a highly speci c mitogen

or vascular endothelial cells. The expression o VEGFis potentiated in tumors in response to hypoxia. Thisprocess is aided by a variety o activated oncogenes andcytokines. VEGF plays a central role in the regulation o vasculogenesis and angiogenesis by inducing endothe-lial cell proli eration, migration, and maturation and byinhibiting their apoptosis.

Anti-phospho-IGF-1R(Tyr1161/Tyr1165/Tyr1166)(Catalogue No. ABE332)

Insulin-like growth actor 1 receptor (IGF-1 receptor, IGF-1R) binds IGF-1, a peptide hormone secreted in responseto growth hormone signaling. IGF-1R, a receptor tyrosinekinase, phosphorylates cytoplasmic signaling proteins,including Akt/mTOR pathway proteins, to promotegrowth and cell survival. IGF-1R is highly expressed in allcell types and tissues and is highly overexpressed in mostmalignant tissues.

VEGF secretion detected inhuman placental villi usingimmunohistochemistrywith Anti-VEGF (1:400, Cat.No.ABS82) and the EMDMillipore IHC Select Detec-tion Kit (Cat. No. DAB050).

Peptide inhibitionassay demonstrating thatAnti-phospho-IGF-1R (1:200,Cat. No.ABE332) pre-incubated with speci c phos-phopeptide blocks detectiono IGF-1R in IGF-1-treatedHEK293 lysates (lane 3),whereas no peptide or incu-bation with non-phosphory-lated peptide does not blockdetection o IGF-1R (lanes 1and 2, respectively).

growth actor receptors are those with tyrosine kinaseactivity, G-protein coupled receptors and those with

serine/threonine kinase activity. Typically, binding o growth actors to receptors on quiescent cells leads tothe activation o intrinsic receptor-associated tyrosinekinase activity and concomitant phosphorylation o tyrosine residues o cytoplasmic proteins.

Featured Products

Extracellular SignalingThe binding o molecules, such as hormones, growth actors, neurotransmitters, and pharmacologicalagents, to the extracellular domains o transmembrane receptors triggers many important signalingevents inside the cell. Transmembrane receptors are some o the most abundant proteins in plasmamembranes. These receptors bind agonists, which elicit a highly speci c and selective response, andantagonists, which induce non-responsiveness by blocking a receptor. Understanding the biologicalactivity associated with receptor-ligand interactions is central to unraveling signaling pathways andthus orms the ramework or research, drug discovery and development programs.
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Anti-EGF(Catalogue No. 07-1432 )

Epidermal growth actor (EGF) has a pro ound e ect onthe di erentiation o speci c cellsin vivo and is a potentmitogenic actor or a variety o cultured cells o bothectodermal and mesodermal origin. EGF binds to the EGFreceptor (EGFR), which leads to cell proli eration, migration,

adhesion and other processes via the MAPK, JNK, Akt andother pathways.

Anti-EGFR(Catalogue No. 05-1047 )

The EGF receptor (EGFR) is the cell-sur ace receptor ormembers o the EGF amily and is activated by bindingspeci c ligands, including the trans orming growth actor(TGF). Upon activation, EGFR homodimerizes, stimulatingits intracellular protein tyrosine kinase activity, leading tocell proli eration. Certain EGFR mutations are associatedwith di erent orms o cancer.

EGF staining o coloncancer tissue using Anti-EGF(1:100, Cat. No.07-1432)and the EMD Millipore IHCSelect HRP-DAB detectionsystem (Cat. No. DAB500).

EGFR (red) expression inA431 cells is demonstratedusing a Cy-3 conjugatedprimary antibody (Cat. No.05-1047 ) via con ocal im-munocytochemistry, withnuclear staining (blue) andactin staining (green).

MILLIPLEXmap EpiQuantEGFR Signaling Pathway Panel(Catalogue No. MPEQMAG-110K )

The ability to quantitatively analyze phosphorylation

status o EGFR (ErbB) amily members, as well as receptor-related intracellular proteins, is necessary or a thoroughunderstanding o this pathway. This 22-plex immunoassayis ideal or absolute quantitation o ErbB pathwayconstituents, including phosphorylation sites on EGFR,

Time course o EGFR pathway protein phosphorylation showspersistent phosphorylation o EGF receptors (A) but moretransient phosphorylation o other pathway proteins (B). Allanalytes, as well as total EGFR, were simultaneously detectedin A431 cells treated with 100 ng/mL EGF. Values are inter-nally normalized utilizing the TAFII68 loading control.

ErbB2, ErbB3, and 16 other receptor related proteins.

It also enables quantitation o total EGFR and a loadingcontrol (TAFII68) simultaneously in the same assay well.The Sample Preparation Kit (Cat. No. MPEQ-SP) is requiredbe ore running any EpiQuant Panel.

[ A n a l y

t e ] ( p M )

Time (min)

10 10 20 30 40 50 60 70

1000

100

10

EGFR (pY1110/1125) HER2 (pY1023)ErbB3 (pY1197/1307) EGFR totalEGFR (pY1069/1092)

A n a l y

t e (

p M )

Time (min)

0.10 10 20 30 40 50 60 70

100

10

1

FRS2 (pY436) Gab2 (pY614)Gab2 (pY584) Gab2 (pY266)Gab1 (pY285/307/317)

A B
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Key Products

FlowCellect EGFR RTKActivation Dual Detection Kit(Catalogue No. FCCS025107)

Harness the power o multiparameter fow cytometry todetect the extent o EGF pathway activation by measuringthe EGFR phosphorylation in relation to the total EGFRexpression in any given cell population. The levels o both

the total and phosphorylated protein can be measuredsimultaneously in the same cell, resulting in a normalizedand accurate measurement o EGFR activation a terstimulation, and o ering more reliable detection o thephospho:total ratio within a mixed population o cells.

Dual parameter analysis o total and phospho-EGFR on A431 cells. Untreated cells stainedwith an isotype control (A) and both pEGFR-Alexa Fluor 488 and Anti-EGFR-PerCP (B)expressed mainly unphosphorylated EGFR (88.6% o cells). However, once A431cells werestimulated with 100 ng/mL EGF, the percentage o cells with phosphorylated EGFR increased

rom 5% to 98% (compare double positive cells in (B) and (D)). Target speci city o phosphory-lation was con rmed by simultaneous measurement o both total and phospho EGFR. A431stimulated cells showed no activity when stained with an isotype control (C).

Description Catalogue No.

Antibodies

Anti-EGF 07-1432

Anti-EGFR (cytoplasmic domain) 05-1047

Anti-EGFR, polyclonal 06-847

Anti-FGFR-4 07-2112

Anti-phospho-IGF-1R (Tyr1161/Tyr1165/Tyr1166) ABE332

Anti-TGF- Receptor, type I 06-1086

Anti-VEGF ABS82

Kits and Assays

FlowCellect EGFR RTK Activation Dual Detection Kit FCCS025107

MILLIPLEXMAPEpiQuant EGFR Signaling Pathway Panel MPEQMAG-110K

MILLIPLEXMAPTotal EGF Receptor MAPmate Assay 46-606

MILLIPLEXMAPHuman Angiogenesis/Growth Factor Panel HAG1MAG-12K

Proteins

PDGFR, active 14-467

Trans orming Growth Factor-, recombinant human GF022

A. Isotype Control (No EGF stimulation) B. Total and pEGFR (No EGF st imulat ion)

0.5%

1.5%

98%

0%

5.5%

0.5% 5.4%

88.6%

C. Isotype Control (EGF stimulation) D. Total and p EGFR (EGF stimulation)

0.2%

0.6% 98.2%

1%

0% 0.3%

0.7%99%

A

C

B

D
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Featured Products

Me N

O

O O

OH

NH

NH

Cytokines and ChemokinesCytokines and chemokines encompass large and diverse

amilies o immunomodulating polypeptide regulatorsthat are involved in mediating and regulating immunity,infammation, and hematopoiesis. Secreted by speci ccells in response to immune stimulus, these proteinsmediate local intercellular communication and di er

rom hormones with respect to circulating concentra-tions and distribution. Unlike growth actor receptors,

cytokine receptors generally lack identi able catalyticactivity. Cytokine receptors contain multiple cysteineresidues and a conserved amino acid moti WSXWS(Trp-Ser-X-Trp-Ser) that unctions in the recognitionand binding o the ligand. Understanding cytokine andchemokine signaling can shed light on disease states,

such as allergic reactions, infammatory bowel disease(IBD), sepsis, and cancer.

Anti-Interleukin 17 Receptor (IL-17R)(Catalogue No. 06-1071 )

Interleukin-17 receptor (IL-17R) is a cytokine receptorthat speci cally binds to IL-17B and IL-17E, but does notbind to IL-17 or IL-17C. The IL-17 amily o proinfam-matory cytokines are produced by activated T cells, andhigh levels o IL-17 cytokines are associated with severalchronic infammatory diseases, including rheumatoidarthritis, psoriasis and multiple sclerosis. Overexpressiono IL-17R is linked to nonrecurrence a ter tamoxi enchemoprophylaxis in hormone receptor-positive breastcancer.

CXCR2 Antagonist, Cpd 19(Catalogue No. 239819 )

This potent antagonist o chemokine receptor CXCR2(IL8R, IC50 = 8 nM) is a cell-permeable, cyclobutenedi-one derivative, ideal or chemical genetics studies o chemokine-activated chemotaxis o neutrophils towardsites o infammation. Overexpression o CXCR2 andits ligand, IL-8, are associated with diseases caused bydysregulated infammation, such as arthritis, asthma, andchronic obstructive pulmonary disease.

Flow cytometry analysis o human PBMCs with 1 gAnti-IL-17 Receptor (Cat.No.06-1071 , A). Westernblot analysis o mouse testislysate probed with Anti-IL-17Receptor (1 g/mL, B). IL-17 Receptor was detectedat ~56 kDa.

Cpd 19 is in the phenol-containing class o CXCR2

antagonists, which is thecompound class that hasbeen the most ruit ulsource o clinical candi-dates. Reported to inhibitCXCR2-mediated chemo-taxis in a CXCR2 expressingcell line (IC50 = 145 nM), itexhibits good stability in hu-man and rat liver microsomalpreparations (>50% remain-ing a ter 30 min at 37C).

A B
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0

0

2000

4000

6000

0 2 5 10 15 30 60 120

M F I

IFN Time Course (minutes)

MILLIPLEXMAP STAT 5-PlexPanel(Catalogue No. 48-610)

Multiple cytokine, hormone and growth actor recep-tors utilize JAK/STAT pathways or signaling. Quanti yingthe relative expression o STAT transcription actors isimportant or understanding the relationship betweenextracellular and nuclear signaling in normal physiologi-cal as well as pathological states including oncogenesis,immune responses and stem cell di erentiation. Thispanel enables the simultaneous detection o multipleSTAT proteins in a single well.

FlowCellect Mouse TH1/TH2Identi cation Kit(Catalogue No. FCIM025137)

FlowCellect kits or intracellular cytokine assays arecompletely optimized and validated or fow cytometerswith blue and red lasers. The kits include all necessaryreagents and bu ers, including a xable viability dye.The FlowCellect Mouse TH1/TH2 Identi cation Kitprovides an easy way to evaluate the pro le o animmune response, by detecting IFN- and IL-4 expressionin mouse TH1 and TH2 CD4+ T-cells.

Increased levels o phosphorylated STAT1, 2, 3, 5 and 6 instimulated cells compared to their unstimulated counterpartsas detected using the MILLIPLEXMAPHuman STAT 5-PlexPanel. Phosphorylated STAT proteins were simultaneouslydetected in HeLa cells treated with 2,000 U/mL IFN or 0, 2,5, 10, 15, 30, 60, and 120 minutes.

Cytokine expression in Th1 and Th2 cells detected usingfow cytometry. Th1- and Th2-di erentiated Cd4+ mouseT cells were stained with the FlowCellect Mouse TH1/TH2Identi cation Kit. Double-positive cells (in the upper rightquadrant o each plot) represent live, CD4+ lymphocytes that

also express IFN (Th1-di erentiated cells, A) or IL-4 (Th2-di erentiated cells, B).

A B

MILLIPLEXMAP STAT Panel:IFN Time Course

Featured Products
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1

Key Products


Antibodies

Anti-IL-16, clone 14.1 MABF29

Anti-Interleukin 10 Receptor (IL-10R) 06-1067

Anti-Interleukin 17 Receptor (IL-17R) 06-1071

Anti-Interleukin 21 (IL-21) 06-1074

Anti-Interleukin 23 (IL-23) p40, clone 2G6 04-1582





Anti-TNF 04-1114

Anti-TNF Receptor MAB3216

Small Molecule Inhibitors

CXCR2 Antagonist, Cpd 19 239819

CXCR4 Antagonist I, AMD3100 239820

CXCR4 Antagonist II 239821

Kits and Assays

FlowCellect Mouse TH1/TH2 Identi cation Kit FCIM025137

MILLIPLEXMAPHuman Soluble Cytokine Receptor Panel HSCRMAG-32K

MILLIPLEXMAPHuman STAT 5-Plex Panel 48-610MAG

MILLIPLEXMAPTGF Signaling Pathway 6-Plex Panel 48-614MAG

Proteins

TNF-, recombinant rat GF046

Interleukin-11, recombinant IL011
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G-Protein CoupledReceptors (GPCRs)GPCRs are one amily o specialized transmembranereceptors proteins that acilitate communication withthe extracellular environment. A receptors main unctionis to recognize and respond to molecules on theextracellular domain and initiate a response or signalingcascade via G protein signaling rom the intracellular

domain. GPCR-associated signaling pathways controlnumerous essential unctions in all tissues and areubiquitous throughout the animal kingdom. EMDMillipore provides a wide range o products oridenti cation and quanti cation o sur ace expressionlevels and activity levels o GPCRs.

Anti-GPR177, clone YJ5(Catalogue No. MABS87)

Wnt amily proteins are secreted signaling proteins thatregulate di erentiation and development, and requentlyregulate gene expression in tissues at sites distant romthe point o secretion. GPR177 (WLS, EVI), the mouseortholog o Drosophila Wntless, is a G protein-coupledreceptor that binds to Wnt and helps mediate itslong-range e ects. GPR177 is essential or the pattern-ing o the anterior-posterior axis during mammaliandevelopment and is activated by-catenin and LEF/TCF-dependent transcription. Upon GPR177 activation,Wnt binds to and modi es the subcellular distributiono GPR177, leading to a eedback regulatory mechanisminvolving Wnt expression and signaling.

GPR30 Agonist, G-1(Catalogue No. 371705 )

Although estrogen is best known or binding to nuclearreceptors to regulate gene expression, it also binds toGPR30, a GPCR localized to the endoplasmic reticulum,to mediate rapid cellular responses to injury via the PKApathway. Use this high-a nity agonist or GPR30 tostudy the clinically important nongenomic roles o es-trogen. G-1 speci cally competes with estrogen bindingto GPR30 (Ki = 11 nM), but does not bind the classicalnuclear-residing estrogen receptors, ER and Er.

GPR177 detected on subcellular membranes in HeLa cellstrans ected with human GPR177 using immunocytochemistrywith Anti-GPR177, clone YJ5 (1:50, Cat. No.MABS87, A).GRP177 was visualized using a Goat Anti-Mouse IgG second-ary antibody conjugated to Alexa Fluor 594 dye (Red). Nucleiare stained with DAPI (Blue). (Data courtesy o Pro . D.M.

Virshup, Program in Cancer and Stem Cell Biology, Duke-NUSGraduate Medical School.) Western blot analysis o HeLacell lysate probed with Anti-GPR177, clone YJ5 (1:5,000, B).GPR177 was detected at ~62 kDa.

A cell-permeable, nonsteroidal, dihydroquinoline compound,G-1 has been shown to stimulate GPR30-mediated cellularPI3K activation and calcium mobilization (EC50 = 2 nM inCOS7 cells) and inhibit migration o SKBr3 and MCF-7 cellstoward chemoattractants (IC50 = 0.7 and 1.6 nM, respectively).

O

O

OH

HNH

Br

CH3

Featured Products

A B
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Key Products

ChemiSCREEN CXCR4Membrane Preparation(Catalogue No. HTS004M)

C-X-C chemokine receptor type 4 (CXCR4) transduces asignal by increasing intracellular calcium and is vital orcell tra cking during development. Stromal-derived ac-tor 1 (SDF-1) binds to CXCR4 expressed on hematopoi-etic and lymphopoietic cells, and directs their tra ckingto sites o infammation. CXCR4 is expressed on severaltumor cell lineages and may direct metastasis to sites o SDF-1 expression. ChemiSCREEN CXCR4 membranepreparations are made rom stable cell lines that expresshigh levels o recombinant CXCR4 and are ideal tools orscreening or CXCR4 and SDF-1 antagonists.

Competitive radioligandbinding assay results usingthe ChemiSCREEN CXCR4

membrane preparation and125I-labeled SDF-1 and un-labeled SDF-1 demonstratetotal binding (TB), nonspeci cbinding (NSB) and speci cbinding (SB).


Antibodies

Anti-GPR177, clone YJ5 MABS87

Anti-GRK 2/3 (ARK 1/2) 05-465


GPR30 Agonist, G-1 371705

S1P1 Receptor Agonist, SEW2871 567733

SB 290157 559410

Kits and AssaysChemiSCREEN CXCR4 Membrane Preparation HTS004M

FlowCellect Chemokine Receptor CXCR4 Sur ace Kit FCXR400423

90000

80000

70000

60000

5000040000

30000

20000

10000

00.0 0.5 1.0 1.5 2.0 2.5

NSB TB

[125 I] - SDF1- (nM)

[ 1 2 5 I ] -

S D F 1 -

B o u n

d

( c p m

)

SB
http://www.millipore.com/catalogue/item/HTS004M?cid=BIOS-S-EPDF-1111-1208-RChttp://www.millipore.com/catalogue/item/MABS87?cid=BIOS-S-EPDF-1111-1208-RChttp://www.millipore.com/catalogue/item/05-465?cid=BIOS-S-EPDF-1111-1208-RChttp://www.millipore.com/catalogue/item/371705?cid=BIOS-S-EPDF-1111-1208-RChttp://www.millipore.com/catalogue/item/567733?cid=BIOS-S-EPDF-1111-1208-RChttp://www.millipore.com/catalogue/item/559410?cid=BIOS-S-EPDF-1111-1208-RChttp://www.millipore.com/catalogue/item/HTS004M?cid=BIOS-S-EPDF-1111-1208-RChttp://www.millipore.com/catalogue/item/FCXR400423?cid=BIOS-S-EPDF-1111-1208-RChttp://www.millipore.com/catalogue/item/FCXR400423?cid=BIOS-S-EPDF-1111-1208-RChttp://www.millipore.com/catalogue/item/HTS004M?cid=BIOS-S-EPDF-1111-1208-RChttp://www.millipore.com/catalogue/item/HTS004M?cid=BIOS-S-EPDF-1111-1208-RChttp://www.millipore.com/catalogue/item/559410?cid=BIOS-S-EPDF-1111-1208-RChttp://www.millipore.com/catalogue/item/567733?cid=BIOS-S-EPDF-1111-1208-RChttp://www.millipore.com/catalogue/item/371705?cid=BIOS-S-EPDF-1111-1208-RChttp://www.millipore.com/catalogue/item/05-465?cid=BIOS-S-EPDF-1111-1208-RChttp://www.millipore.com/catalogue/item/MABS87?cid=BIOS-S-EPDF-1111-1208-RC


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Featured Products

Ion ChannelsIon channels, the ion-permeable pores in lipid bilayers,constitute a large amily o pore- orming transmembraneproteins. They open and close in response to speci cstimuli and acilitate the transport o charged moleculesand control the voltage across the plasma membraneby acilitating the fow o ions down an electrochemical

gradient. Ion channels enable rapid signaling in electri-

cally excitable cells, such as neurons. In the open con-ormation, ions are tightly bound by the channel, which

discriminates between sizes and charges o the pervadingmolecules. The con ormational change between closedand open state (gating) is controlled by external modula-tors such as ligand binding, voltage, second messengers,

and G-proteins.

Anti-Calcium Channel, Voltage-Gated 1C (Cav1.2)(Catalogue No. AB5156)

Voltage-sensitive calcium channels mediate the infux o calcium ions into the cell upon membrane depolarizationand are involved in processes such as muscle contraction,neurotransmitter release, cell motility, and cell death.The iso orm-1c gives rise to L-type (long-lasting)calcium currents. L-type channels are sensitive todihydropyridines, which are requently used to treathypertension. Expression is seen in brain, heart, jejunum,ovary, pancreatic-cells, and vascular smooth muscle.

Anti-K+ /Cl- Cotransporter (KCC2),clone N1/12(Catalogue No. MABN88)

KCC2, a cation-chloride cotransporter, is proposed to actas the main chloride extruder to promote ast hyperpo-larizing postsynaptic inhibition in the brain. KCC2 is ex-pressed at high levels in neurons throughout the nervoussystem and immunofuorescence shows that the proteinis localized at inhibitory synapses o the spinal cord.Studies in mice have shown that KCC2 reduces GABAsinhibitory signaling, resulting in motor de ects, epilepsy,and anxiety-like behavior.

Immunocytochemical analysis o the rat cerebellum usingAnti-Calcium Channel, Voltage Gated 1C. This antibodypositively stains Purkinje cells (red). Photo courtesy o Dr. W.Hartig and J. Grosche, Leipzig University, IZKF.

KCC2 is expressed in the neuronal membranes and synapseso the rat brain. Immunohistochemistry analysis o para n-embedded rat hypothalamus (A) and cortex area A-3 (B)using Anti-K+/Cl-Cotransporter (KCC2), clone N1/12 (1:300,Cat. No.MABN88). Reactivity was detected using the IHCSelect Detection Kit (Cat. No. DAB050).

A B
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Key Products

PrecisION Membrane PreparationRecombinant hERG PotassiumIon Channel(Catalogue No. CYL4039)

The human ether-a-go-go-related gene (hERG) encodes avoltage-gated potassium channel which mediates cardiacaction potential repolarization in the mammalian heart.Many drugs and drug candidates can block the hERGchannel, causing cardiotoxicity, making hERG screeninga critical preclinical study. This membrane preparationis made rom the PrecisION recombinant hERG ionchannel stable cell line; like all PrecisION cell lines, itis biophysically and pharmacologically validated usingelectrophysiology.

Competition binding or

hERG membrane preparationversus wild-type HEK293membrane preparations us-ing [3H]-astemizole, a knownhERG antagonist.


Antibodies

Anti-Acid Sensitive Ion Channel 1 AB5674P

Anti-Cav1.2 Calcium Channel, Voltage Gated 1C AB5156

Anti-Cav1.2 Calcium Channel, clone L57/46 MAB13170

Anti-K+/Cl- Cotransporter (KCC2), clone N1/12 MABN88

Anti-Potassium Channel Kv1.2, clone K14/16 MABN77

Anti-Sodium channel Nav1.7, clone N68/6 MABN41Kits and Assays

PrecisION Membrane Preparation Recombinant hERG Potassium Ion Channel CYL4039

10 g/well WT 10 g/well

[ 3 H ] - A s t e m

i z o l e

b o u n

d

( c p m

)

[Astemizole] Log M

700

600

500

400

300

200

100

0-12 -11 -10 -9 -8 -7 -6 -5
http://www.millipore.com/catalogue/item/CYL4039?cid=BIOS-S-EPDF-1111-1208-RChttp://www.millipore.com/catalogue/item/AB5674P?cid=BIOS-S-EPDF-1111-1208-RChttp://www.millipore.com/catalogue/item/AB5156?cid=BIOS-S-EPDF-1111-1208-RChttp://www.millipore.com/catalogue/item/MAB13170?cid=BIOS-S-EPDF-1111-1208-RChttp://www.millipore.com/catalogue/item/MABN88?cid=BIOS-S-EPDF-1111-1208-RChttp://www.millipore.com/catalogue/item/MABN77?cid=BIOS-S-EPDF-1111-1208-RChttp://www.millipore.com/catalogue/item/MABN41?cid=BIOS-S-EPDF-1111-1208-RChttp://www.millipore.com/catalogue/item/CYL4039?cid=BIOS-S-EPDF-1111-1208-RChttp://www.millipore.com/catalogue/item/CYL4039?cid=BIOS-S-EPDF-1111-1208-RChttp://www.millipore.com/catalogue/item/CYL4039?cid=BIOS-S-EPDF-1111-1208-RChttp://www.millipore.com/catalogue/item/MABN41?cid=BIOS-S-EPDF-1111-1208-RChttp://www.millipore.com/catalogue/item/MABN77?cid=BIOS-S-EPDF-1111-1208-RChttp://www.millipore.com/catalogue/item/MABN88?cid=BIOS-S-EPDF-1111-1208-RChttp://www.millipore.com/catalogue/item/MAB13170?cid=BIOS-S-EPDF-1111-1208-RChttp://www.millipore.com/catalogue/item/AB5156?cid=BIOS-S-EPDF-1111-1208-RChttp://www.millipore.com/catalogue/item/AB5674P?cid=BIOS-S-EPDF-1111-1208-RC


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Many proteins, like insulin, are synthesized as inac-

tive precursors and must be proteolytically cleaved oractivation. However, there are several other modi cationssuch as acetylation, methylation, phosphorylation, and

4G10 Platinum,Anti-Phosphotyrosine(Catalogue No. 05-1050)

Well known or its sensitive recognition o multiplephosphotyrosines on numerous substrates, 4G10 hasbeen validated by thousands o researchers in virtuallyevery application and tyrosine target. To improve 4G10

urther, we pooled it with the next most highly regardedphosphotyrosine antibody, clone PY20, to make 4G10Platinum, enabling more sensitive detection o moresubstrates than 4G10 alone.

Con ocal immunofuorescence using 4G10 Platinum Anti-phosphotyrosine o EGF-treated A431 cells (right) versusuntreated (le t) demonstrate activation via phosphorylation(green). Nuclear staining was achieved using DAPI (blue).

ubiquitination that impact protein unction and activity.

As a result, the analysis o proteins and their post-trans-lational modi cations is particularly important or thestudy o normal and disease-associated processes.

Anti-Ubiquitin K11 Linkage,

clone 2A3/2E6(Catalogue No. MABS107)

Ubiquitin is a small protein that can be covalently at-tached to proteins either as a monomer or as a polymer.Lysine11 (K11)-linked ubiquitin chains regulate mitoticprotein degradation by acting as a speci c substrate orthe anaphase-promoting complex (APC), which has E2and E3 ubiquitin ligase activity. Antibodies speci c tothe K11-linkage have shown increased ormation o K11chains in mitotic human cells in the presence o APCsubstrate degradation.

Immunocytochemistry analysis o HeLa (A) and A431 (B) cellsusing 1:500 Anti-Ubiquitin K11 linkage, clone 2A3/2E6 (Cat. No.MABS107, Red). Actin laments have been labeled with Alexa

Fluor 488 dye-Phalloidin (green). Anti-Ubiquitin K11 stainsboth the cytosol and nucleus (with intense staining on nucleus).

Featured Products

Cytosolic SignalingCytosolic signaling proteins play crucial roles in cell cycling, targeted proteolysis, protein tra cking,cytoskeletal organization, and gene expression. Mutations in or misregulation o these proteinsare associated with initiation, promotion, and progression o various diseases including cancer,diabetes and rheumatoid arthritis. Cytosolic signaling is dominated by protein phosphorylation anddephosphorylation by protein kinases and protein phosphatases, which represent the most commontargets o therapeutics, accounting or over 25% o drug discovery programs worldwide. A leaderin cell signaling, EMD Millipore is building the most comprehensive selection o tools or studies o phosphorylation and other post-translational modi cations.

A

A

B

B

Post-Translational Modi cations


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Anti-Ubiquitin, Lys48-Speci c,clone Apu2(Catalogue No. 05-1307)

Polyubiquitin chains linked through the Lys48 residue o ubiquitin are most commonly associated with proteinstargeted or proteosomal degradation. Misregulationo protein metabolism can lead to unchecked proli era-tion and tumorigenesis. Visualize protein turnover withthis robust anti-ubiquitin (Lys48-speci c) monoclonalantibody.

Anti-phospho-Estrogen Receptor(ER) (Ser305), clone 124.9.4(Catalogue No. 05-922R)

Resistance to the ER-binding drug, tamoxi en, is one o

the major challenges in breast cancer treatment. Phos-phorylation o ER at serine 305 (ERS305-P) by proteinkinase A (PKA) leads to an activation o ER and to tran-scription o ER-responsive genes a ter tamoxi en treat-ment, leading to tamoxi en resistance1,2. Clinical studies

Con ocal IF using Cat. No.05-1307 shows Lys48-linkedubiquitin, marking proteins

or degradation. Ubiquitinsignal (red, le t panel) isstrongest in the nucleus(blue, right panel).

Immunohistochemistryanalysis o ductal carcinomausing phospho-ER (Ser305),clone 124.9.4 (1:200).

Nuclear reactivity (as seenhere in both images) wasonly observed when the can-cer case was ully involvedwith ER only and with a highproli eration rate combinedwith a high S-phase and DNAindex.

also show that ERS305-P may be a good biomarker to

identi y patients unlikely to respond to tamoxi en3,4

.Our extensively validated antibody or ERS305-Pspeci cally reacts with breast cancer tissue rom samples

rom ER-positive patients as shown by immunohisto-chemistry.

1. Zwart W et al. PKA-induced resistance to tamoxi en is associated withan altered orientation o ERalpha towards co-activator SRC-1. EMBO J.2007 Aug 8;26(15):3534-44.

2. Wang RA et al. P21-activated kinase-1 phosphorylates and transacti-vates estrogen receptor-alpha and promotes hyperplasia in mammaryepithelium. EMBO J. 2002 Oct 15;21(20):5437-47.

3. Holm C et al. Phosphorylation o the oestrogen receptor alpha at serine305 and prediction o tamoxi en resistance in breast cancer. J Pathol.2009 Feb;217(3):372-9.

4. Kok M et al. PKA-induced phosphorylation o ER at serine 305 andhigh PAK1 levels is associated with sensitivity to tamoxi en in ER-positive breast cancer. Breast Cancer Res Treat. 2011 Jan;125(1):1-12.

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Nitrotyrosine ELISA(Catalogue No. 17-376)

Reactive oxygen species (ROS), such as nitric oxide

(NO

), superoxide (O2

), peroxynitrite (ONOO-

), and hydroxylradical (OH-) can cause nitration o tyrosine residues. The ni-trotyrosine assay kit with chemiluminescence detection is acompetitive ELISA or quanti ying tyrosine nitration. The kitincludes all required reagents, including white, high-binding96-well plates, nitrated BSA standard, anti-nitrotyrosine an-tibody, LumiGLO detection substrate, and wash bu ers. Theassay has a wide dynamic range and high precision, makingit a valuable tool or studying oxidative stress.

MILLIPLEXMAP EpiQuant

Human Receptor SignalingPathway Panel(Catalogue No. MPEQMAG-104K)

Receptor phosphorylation is one o the key steps in ini-tiating signal transduction rom the membrane. Use this27-plex immunoassay based on EpiQuant technology

or absolute quantitation o 11 receptor signaling-relatedprotein targets as well as the degree o phosphorylationat speci c sites on these proteins. These total and phos-phorylated proteins include HER2, HER3, VEGFR1 and

VEGFR2, among others. We have validated this panel orthe analysis o various stimulated tissue culture lysates.

The linear range o the nitrotyrosine ELISA encompasses twoorders o magnitude.

Serum starvation decreases receptor phosphorylation (but notPTEN levels) in HEK293 and Kasumi-3 cellular lysates. Phos-phorylated proteins were measured in parallel in lysates romserum-starved and serum- ed cells. Total protein levels weresimultaneously assessed in the same well or each o the listedphosphorylation targets and used to normalize expression values.


AntibodiesAnti-AMPylated Threonine 09-890

Anti-dimethyl-Arginine, asymmetric (ASYM25) 09-814

Anti-modi ed Citrulline 07-2168

Anti-O-GlcNAc, clone 1F5.D6(14) 05-1246

Anti-Sul otyrosine, Clone Sul o-1C-A2 05-1100

Focal Adhesion Pathway Explorer Antibody MiniPack 15-113

mTOR Phosphorylation Pathway Explorer Antibody MiniPack 15-105

Kits and Assays

MILLIPLEXMAPEpiQuant Phosphothreonine Cell Signaling Panel 1 MPEQMAG-103K

MILLIPLEXMAPHeat Shock Protein 5 Plex 48-615MAG

MILLIPLEXMAPMulti-Pathway 9-Plex 48-680MAG

n o c o m

p e t i t o r

Nitrated BSA (g/mL)

C P S

x 1 0 0

, 0 0 0

0.1 1.0 10 100 1000 10000

80

70

60

90

50

40

30

20

10

0

Featured Products

Key Products

P h o s p

h o p r o

t e i n C o n c e n t r a

t i o n

( p

M )

1000

800

600

400

200

0

L y n ( p Y 3

9 7 )

R E T (

p Y 8 2

6 )

R E T (

p Y 1 0

6 2 / 1 6

9 0 / 1 6

9 6 )

H E R 2

( p Y 1

1 1 2 / 1

1 3 9 )

H E R 2

( p Y 1

1 9 6 / 1

2 2 1 / 1

2 2 2 )

H E R 2

( p Y 1

0 2 3 )

S y k (

p Y 3 2

3 )

P T E N

T O T A

L

HEK293: serum starvedHEK293: serum fedKasumi-3: serum starvedKasumi-3: serum fed
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Kinases, Phosphatases andTheir SubstratesPhosphate groups are key posttranslational modi cationsto proteins in signaling pathways. Protein phosphoryla-tion and dephosphorylation regulate normal physi-ological and pathological processes that contribute tocomplications, including cancer, central nervous systempathologies, diabetes, pain, infammation, autoimmune

IRS1 transmits insulin signals via metabolic and mi-togenic pathways. IRS1 is heavily phosphorylated onboth serine and tyrosine residues. These phosphoryl-

ated tyrosines enable IRS to act as a docking proteinthat binds SH2 domains o such proteins as PI3 Kinase(phosphatidylinositol 3-kinase) and GRB2, resulting inactivation. Over expression and phosphorylation o serineis associated with insulin resistance and breast cancer.Ser302 is phosphorylated ollowing insulin stimulation.Ser307, phosphorylated by JNK and IKK, is a key regula-tory site that appears to disrupt the IRS1/IR interactionand inhibits insulin-mediated activation o the PI3 kinaseand MAPK pathways, and Ser636/639 is known to bephosphorylated by p70S6K downstream o mTOR in anegative eedback loop.

diseases, and cardiotoxicity. There ore, the responsiblemodi ying enzymes (kinases and phosphatases) and theirsubstrates have been the ocus o therapeutic modula-tion, chemical genetics experiments and quantitativedetection or the elucidation o signaling networks anddynamics.

Insulin Receptor Substrate 1 (IRS1) Antibodies

Unphosphorylated IRS1 detection:Anti-IRS1, clone 58-10C-31 (Catalogue No. AB5156)

Phosphorylated IRS1 detection:Anti-phospho-IRS1 (Ser632), clone 5.3.3 (Catalogue No. 05-1568 )Anti-phospho IRS1 (Ser522), clone 17.5.2 (Catalogue No. 05-1921 )Anti-phospho IRS1 (Ser318), clone 3.1.1 (Catalogue No. MABS138)

A. Western blot analysis o 3T3/A31 (lane 1),3T3/L1 (lane

2), L6 (lane 3), and MCF7(lane 4) lysates probed withAnti-IRS1, clone 58-10C-31(1:1000) showed IRS1 as aband at ~160 kDa.

B - D. IR/IRS1 trans ectedCHO cells with (+) andwithout (-) phosphataseinhibitors calyculin A andokadaic acid. Lysates weresubjected to Western blot-ting and probed with: (B)Anti-phospho-IRS1 (Ser632)(Cat. No.05-1568), (C) Anti-phospho IRS1 (Ser522), clone17.5.2 (Cat. No.05-1921),or (D) Anti-phospho IRS1(Ser318), clone 3.1.1 (Cat.No.MABS138). Phospho-IRS1 was detected at ~160kDa.

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A

C

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D

(kDa)260

160

11080

60

504030

20

15

10

1 2 3 4

260

160

110

80

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50

40

30

20

15

Cal/Oka - +

(kDa)260

160110

80

60

5040

30

20

1510

Cal/Oka - +

(kDa)260

160

110

80

60

50

40

30

20

15

Cal/Oka - +
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Anti-Calcineurin(Catalogue No. 07-1490 )

Calcineurin/PP2B is a serine/threonine protein phos-

phatase involved in a variety o calcium-mediated cellu-lar responses. Calcineurin/PP2B has been linked to Pmk1MAP kinase and phosphoinositide (PI) pathways, as wellas to members o the small GTPase Rab/Ypt amily andType II myosin. Furthermore, calcineurin helps transduceextracellular signals to the nucleus by dephosphorylatingmembers o the NFAT amily o transcription actors.

MILLIPLEXMAP PhosphorylatedAkt/mTOR 11-Plex Panel(Catalogue No. 48-611MAG )

As nearly all o the players in the Akt/mTOR signalingpathway are coordinately regulated by phosphorylation,understanding the role o this pathway in normal physi-ological processes and in diseases such as cancer anddiabetes requires the ability to simultaneously measurephosphorylation status o multiple protein targets. TheMILLIPLEXMAPAkt/mTOR 11-plex Panel has been suc-cess ully used to simultaneously quanti y multiple path-way proteins in cancer cell lines (HepG2, HEK293, andMCF7) as well as in human breast cancer tissue samples.

InhibitorSelect 96-Well TyrosineKinase and Phosphatase InhibitorLibrary IV (Catalogue No. 539747 )

This library consists o 83 pharmacologically active,well-documented, cell-permeable, potent and reversibleprotein kinase and phosphatase inhibitors; the major-ity o kinase inhibitors are ATP-competitive. Use thislibrary to advance target identi cation in drug discovery,biochemical pathway analysis, screening new proteinkinases/phosphatases, high content screening and more.

Calcineurin immunohisto-chemistry reveals morphologyo para n-embedded humanvascular tissue. Tissue waspretreated with citrate pH 6

or antigen retrieval.

(A) E ect o inhibitors onphosphorylation o Akt/mTORpathway proteins in HepG2cells. Cells were pre-treatedwith 0.1 M wortmannin, 0.1M rapamycin, 10 M U0126(MEK1/2 inhibitor), 50 MLY-294002 (PI3K inhibitor),or Ro-31-8220 (PKC andGSK3/ inhibitor) or 30minutes prior to the additiono 10 g/mL insulin or 15minutes.

(B) Validation by immuno-precipitation (IP) /Westernblotting. IP o phosphopro-teins in cell lines treatedwith either insulin or IGF1was per ormed with capturebeads and proteins detectedby Western blotting withthe biotinylated detectionantibodies.

Kinases and phosphatasesrom many enzyme amilies

are inhibited by structurallydiverse small molecules inInhibitorSelect 96-WellTyrosine Kinase and Phospha-tase Inhibitor Library IV.

Enzyme Family Number o Inhibitorsin Library

Receptor Tyrosine Kinase (RTK) 37

Tyrosine Kinase (TK) 27

Tyrosine Kinase-like (TKL) 6

PKA, PKG, PKC amilies (AGC) 1

CDK, MAPK, GSK-3, CLK amilies (CMGC) 3

Receptor Tyrosine Phosphatase (RTP) 1

Tyrosine Phosphatase (TP) 8

% C

o n t r o l

S i g n a l

Phosphorylated Protein

Wor tm an ni n R ap am yc in U 01 26 LY- 29 40 02 R o- 31 -8 22 0

p 7 0 S 6 K ( T h r 4

2 4 )

I R S 1 ( S e r 3

1 2 )

G S K 3

( S e r

2 1 )

I G F 1 R ( T y r

1 1 3 5 / T y r

1 1 3 6 )

G S K 3 ( S e r 9

)

A k t ( S e r

4 7 3 )

P T E N ( S E R 3 8 0 )

I R ( T y r

1 1 6 2 / T y r

1 1 6 3 )

R P S 6 ( S e r 2

3 5 / S e r

2 3 6 )

T S C 2 ( S e r

9 3 9 )

m T O R ( S e r 2

4 4 8 )

150

125

100

75

50

25

0

pp70S6K

pAkt

NT Ins NT IGF NT IGFNT IGF NT SerumNT Ins

pIRS1

pPTEN

pGSK3a

pIR

pIGF1R

pRPS6

pGSK3b

pTSC2 pmTOR

(HepG2) (HEK293) (HEK293) (HepG2) (HEK293) (HEK293)

NT EGF NT IGFNT IGF NT IGFNT IGF(A431) (HEK293) (MCF-7) (HEK293) (MCF-7)

A

B

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Antibodies

Akt/mTOR/S6K Pathway Explorer Antibody MiniPack 15-104

Anti-Calcineurin 07-1490

Anti-IRS1, Alexa Fluor 488 conjugated 16-257

Anti-IRS1, clone 58-10C-31 05-784R

Anti-IRS-2, clone 9.5.2 MABS15

Anti-MAPK 1/2 ABS44

Anti-mouse Tks4 09-260

Anti-phospho IRS1 (Ser318), clone 3.1.1 MABS138

Anti-phospho IRS1 (Ser522), clone 17.5.2 05-1921

Anti-phospho-IRS1 (Ser632), clone 5.3.3 05-1568

Anti-phospho-p38 (Thr180/Tyr182), clone 6E5.2 MABS64


Akt Inhibitor XV, Isozyme Selective 124034

Aurora Kinase Inhibitor VI 189410

InhibitorSelect 96-Well Tyrosine Kinase and Phosphatase Inhibitor Library IV 539747

JNK Inhibitor III, SR-3306 420147

SB 203580 559389

U0126 662005

Y-27632 688000

Kits and Assays

FlowCellect Multi-STAT Activation Pro ling Kit FCCS025550

FlowCellect PI3K/MAPK Dual Pathway Activation and Cancer Marker Detection kit FCCS025100

FlowCellect PI3 Kinase-mTOR Signaling Cascade Kit FCCS025210

MILLIPLEXMAP10-Plex MAPK/SAPK Signaling Kit Phosphoprotein 48-660MAGMILLIPLEXMAPPhosphorylated Akt/mTOR 11-Plex Panel 48-611MAG

Proteins

AMPK (1, 1, 1), active 14-840

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G-Proteins and SecondMessengersLigand binding o G protein-coupled receptors (GPCRs)induces a con ormational change allowing the receptorto unction as a switch, alternating between an inactiveguanosine diphosphate (GDP)- and active guanosinetriphosphate (GTP)-bound states. These di erent states

regulate signal transducing molecules, such as secondarymessengers and kinases, that ultimately impact down-stream cellular processes. The disruption o G-proteinsignals have been implicated in several diseases such as

diabetes, cardiovascular de ects and certain ormso cancer.

Second messengers are molecules that relay signalsreceived and processed by cell sur ace receptors.

They ampli y these signals to cause a biological e ect.Commonly recognized second messengers include cAMP,cGMP, IP3, DAG, and nitric oxide.

Anti-Ras, pan(Catalogue No. 05-1071 )

The product o RAS, the rst human oncogene identi ed,is a small G-protein that activates the Erk/MAPK kinasepathway by activating Ra , and also activates PI3 Kinase(PI3K) and RalGDS. In its oncogenic, mutated state, Rasis unable to hydrolyze GTP to GDP, thus staying in anactive state. Mutations in Ras have been ound in a largepercentage o all human cancers.

Anti-cAMP(Catalogue No. 07-1497 )

Cyclic adenosine monophosphate (cAMP) is an intracel-lular product o ATP catalysis by adenylate cyclase in adownstream response to GPCR activation by extracellularligands. cAMP in turn activates intracellular kinases, thusacting as a second messenger or signal transductionacross the cell membrane. For example, cAMP regulatesthe e ects o glucagon, adrenaline and calcium fuxthrough ion channels. Since it is a ected directly byGPCR activation, cAMP is can be used to monitor GPCRsin the discovery o GPCR-modulating drugs.

Immunohistochemistry

staining o strati ed humansquamous epithelium using Anti-Ras (Cat. No.05-1071)and the IHC Select Detec-tion System.

cAMP detected in para n-embedded rat cerebellumtissue using immunohis-tochemical staining withAnti-cAMP (1:100, Cat.No. 07-1597). Tissue wasprepared using heat-inducedepitope retrieval in citratebu er, pH 6.0. Reactivitywas detected using the IHCSelect Detection Kit (Cat.No. DAB050).

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Rac1 Inhibitor(Catalogue No. 553502 )

Rac1 is a small G protein that signals to the cytoskeletonto modulate cell polarity, adhesion and motility in orderto regulate cell cycle, cell migration and epithelial di -

erentiation. This Rac1 inhibitor speci cally and reversiblyinhibits Rac1 GDP/GTP exchange activity by inter ering

with binding by the Rac-speci c GEFs (guanine nucleo-tide exchange actors) Trio and Tiam1 (IC50 ~50 M).

Ras Activation ELISA Kit(Catalogue No. 17-497 )

The Ras amily o G proteins are GDP/GTP-regulated bi-nary switches that can control cell growth, proli eration,di erentiation, or survival. Because it is a key regulatorin several tumor types, Ras has been a popular target

or cancer research and anti-cancer therapeutics or thepast two decades or both academic and pharmaceuti-cal research. This kit exploits the speci c binding o Ra to activated, GTP-bound Ras. A nity puri cation usingagarose-conjugated GST-Ras binding domain (GST-RBD)o Ra is ollowed by anti-Ras ELISA.

Rac1/Cdc42 Activation Assay Kit(Catalogue No. 17-441 )

Rac1 and Cdc42 are small G-proteins that modulatecytoskeletal organization to a ect cell migration, polarity,growth and other processes. This kit e ectively detectsRac1 and Cdc42 activity in cell lysates. This assay usesthe downstream e ector o Rac1/Cdc42, p21-activatedprotein kinase (PAK1), to isolate the active, GTP-boundRac1/Cdc42 rom the sample. The p21 binding domain(PBD) o PAK1 is expressed as a GST- usion protein andcoupled to agarose beads. A ter precipitation, activatedRac1/Cdc42 can be detected by Western blotting.

EGF-stimulated HeLa celllysates show elevated levelso activated Ras comparedwith the basal levels inunstimulated samples.

Rac1 is activated by GTPbut not GDP in 3T3/A31and HeLa cells. Lysates wereincubated with either GDPor GTPS. Activated Rac1 orCdc42 were isolated usingthe activation assay kit (Cat.No.17-441 ) and detected byWestern blotting with Anti-Rac1 (le t) or Anti-Cdc42(right).

A cell-permeable pyrimidinecompound, Rac1 inhibitore ectively blocks Rac1-mediated cellular unctionsin NIH3T3 and PC-3 cells(e ective dose ~50 to100 M).

N

N

N

N

NH

NH

NH2

3 HCI

HeLa Cell Lysate (50 g/well)

A c

t i v a

t i o n

( R L U )

AssayBackground

-EGF +EGF

Unstimulated EGFStimulated

7.E+066.E+065.E+06

4.E+063.E+06

2.E+061.E+060.E+00

kDa

98

62

4938

28

17

kDa

98

62

4938

28

1714

Rac1

Lane 1: 3T3/A31 GDP

Lane 2: 3T3/A31 GTPS

Lane 3: HeLa GDP

Lane 4: HeLa GTPS

Cdc42

1 2 3 4 1 2 3 4
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Antibodies

Anti-cAMP 07-1497

Anti-Ras 05-1071


Dynamin Inhibitor I, Dynasore 324410

InSolution Rac1 Inhibitor 553508

Rac1 Inhibitor 553502

Kits and Assays

Rac1/Cdc42 Activation Assay Kit 17-441

Ras Activation ELISA Kit 17-497

Lipid SignalingBioactive lipids, generated during remodeling o mem-brane lipids by activated lipases, serve as intra- andextracellular mediators in cell signaling. Although theinteraction between a lipid-based messenger and a cellu-lar receptor can mediate downstream signaling cascadessimilar to other ligand-receptor relationships, lipid sign-

aling is unique in that lipids can reely di use throughcellular membranes. Additionally, lipid messengers canneither be stored in vesicles nor do many lipid signalingmolecules circulate reely in solution. As a result, lipidmessengers are o ten synthesized on demand at the siteo action or are bound to lipid carrier proteins in serum.

Anti-phospho-Grb10(Ser501/Ser503)(Catalogue No. 07-1520)

Growth actor receptor-bound protein 10 (Grb10) wasrecently identi ed as substrate o mTOR and there ore adownstream target o lipid signaling via PI3 kinase.It mediates signals rom a number o activated receptortyrosine kinases, growth actor receptors, and intracellu-lar molecules. Grb10 downregulates insulin signaling via

eedback inhibition o the PI3 kinase pathway, potentially

acting as a tumor suppressor.

Phosphorylated Grb10 ismore abundant in insulin-treated broblasts comparedto untreated cells. Westernblot analysis o untreated(lane 1) and insulin-treated(lane 2) mouse embryonic

broblast cell lysates probed

with Anti-phospho-Grb10(Ser501/Ser503) (1 g/mL).This antibody detected Grb10at ~66 kDa.

Key Products

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Anti-Inositol HexakisphosphateKinase 2 (IP6K2)(Catalogue No. 05-1545)

Generally, inositol phosphates are second messengersin the phosphatidylinositol lipid signaling pathway.IP6K2 converts one o these second messengers, inositolhexakisphosphate (InsP6), to diphosphoinositol pen-takisphosphate (InsP7/PP-InsP5). It may also convert1,3,4,5,6-pentakisphosphate (InsP5) to PP-InsP4, a ect-ing the growth suppressive and apoptotic activities o inter eron- in some ovarian cancers.

Anti-Phospholipase A2(Catalogue No. 05-1406)

Phospholipases hydrolyze phosphoglycerides to ormsmaller compounds which o ten serve as critical secondmessengers. Phospholipase A2 (PLA2) hydrolyzes phos-phatidylcholine to produce lysophosphatidylcholine, asecond messenger involved in cell proli eration, adhesion,T-cell activation, and smooth muscle cell contractility.PLA2 iso orms include membrane-associated, Ca2+-independent orms; cytosolic, Ca2+-dependent orms; andsecretory orms.

Western blot analysis o human testis lysate usingAnti-IP6K2 (1 g/mL), HRPgoat Anti-mouse second-ary antibody conjugate, andchemiluminescence detectionrevealed IP6K2, a roughly50 kDa protein.

Cytoplasmic immunoreactivity detected in malignant cells

in colon cancer tissue using Anti-PLA2 (05-1406) and IHCSelect detection with HRP-DAB.


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InhibitorSelect PI 3-K/Akt/mTORSignaling Pathway Inhibitor Panel(Catalogue No. 124031)

Activation o the PI 3-kinase/Akt/mTOR pathwaystimulates cell proli eration and the translation processin response to nutrients and growth actors.Dysregulation o this pathway can lead to a variety o human tumors. A vast majority o solid tumors arereported to contain mutations either in PTEN or in thecatalytic unit o their PI 3-K, resulting in increased

This panel consists o 12 highly potent, well-characterized, selective, andcell-permeable kinase inhibi-tors that target six proteinsin the PI3 Kinase/Akt/mTORpathway.

PI3 Kinase Activity/Inhibitor ELISA(Catalogue No. 17-493)

The discovery o cancer-speci c mutations in the genecoding or PI3K has trans ormed this eld rom an areao basic biochemistry into one o intense interest ortarget validation and drug development. These mutationssingle out class I PI3K as a particularly important con-tributor to oncogenesis. Most human cancers also showa gain o unction in PI3K signaling. Easily evaluate PI3Kactivation and inhibition with this competitive ELISA kit.

Di erential inhibition o PI3 Kinase by iso orm-speci c versusgeneral class I PI3 kinase inhibitors, as determined using thePI3 Kinase ELISA Kit.

enzymatic activity, cell proli eration, cell invasion, andmetastasis. The InhibitorSelect PI 3-K/Akt/mTORSignaling Pathway Inhibitor Panel enables multiparam-eter analysis, assessment o signal ampli cation/ eed-back, and comparison o biological e ects o perturbingdi erent parts o the pathway.

IGF-1

IGF-1R

PKC

Growth Factors

RTKs

JAK1

PI 3-K

LY 294002 PI-103 PI 3-K Inhibitor PI 3-K Inhibitor II PI 3-K Inhibitor IV PI 3-K Inhibitor VIII Wortmannin

PDK1-Akt-Flt Dual Pathway Inhibitor Akt Inhibitor IV, Akt Inhibitor VIII

PDK1-Akt-Flt DualPathway Inhibitor

PI-103, PI 3-K Inhibitor VIII

Ro-31-8220

JAK1GAB1 BCAP

MDM2

MDM2Nucleusp53

HSP90CDC37

Cytokines AG

Cytokine Receptor BCRBCR

4EBP1

elF4E

PDK-1

Ra 1

PI-103 Rapamycin

mTor

p70s6K

PP2A

DNA-PK

SYK

Caspase 9

Caspase Cascade

ERK Pathway

Translation

ProteinSynthesis

p53 Degradation

GlycogenSynthesis

GSK-3

Akt

Selectivity and Proling AgainstPI3 Kinase Isotypes

0

20

40

60

80

100

120

LY294002Wortmannin

PI-103TGX-221

AS-252424

% C

o n t r o l

p110 p110 p110 p110

Featured Products


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Antibodies

Anti-DEPTOR ABS222

Anti-phospho-Akt1 (Tyr326) AB9927

Anti-PI3 Kinase, p110 09-482

Anti-PI3 Kinase, p110, clone 17D7.2 05-1559

Anti-Pro-Insulin C-Peptide, clone C-PEP-01 05-1109


COX-2 Inhibitor II 236012

Fluvastatin, Sodium Salt 344095

Lovastatin, Sodium Salt 438186

Kits and Assays

Human Apo AIV ELISA EZHAP0A4-73K

MILLIPLEXMAPHuman Drug Metabolism Magnetic Bead Panel HDMMAG-19K

MILLIPLEXMAPHuman Oxidative Phosphorylation (OXPHOS) Magnetic Bead Panel H0XPSMAG-16K

MILLIPLEXMAPRat/Mouse Oxidative Phosphorylation (OXPHOS) Magnetic Bead Panel RM0XPSMAG-17K

PI3 Kinase HTRF Screening Assay 33-016

PI3 Kinase HTS HTRF Assay 33-017

MILLIPLEXMAP Fatty AcidOxidation (FAO) Panels 1 and 2(Catalogue Nos. HFA01MAG-11K, HFA02MAG-11K)

Monitoring the FAO pathway (especially the-oxidationpathway) and any potential cellular metabolism changesin the human tissues and cells can reveal mechanismso response to disease states, drug treatments, dietarychanges or genetic mutations. The MILLIPLEXMAPHu-man Fatty Acid Oxidation Panels include key enzymesthat are involved in the-oxidation pathway, and quan-titation o these targets simultaneously in one reactionwell (multiplexing) provides a more accurate snapshot o pathway activity.

Adipogenesis Assay(Catalogue No. ECM950)

Identi ying regulators o adipogenesis, the ormation o adipose tissue, may enable pharmacological preventionor reversal o obesity. This assay analyzes adipogenesisin the classic 3T3-L1 model. Commonly used inducerso adipogenesis, dexamethasone, IBMX and insulin, areincluded in convenient, ready-to-use ormulations.Staining reagents provided enable quantitative charac-terization o adipogenesis induction or inhibition.

Multiplex analysis o human cell lysates and tissue extract with MILLIPLEXMAPHumanFatty Acid Oxidation Magnetic Bead Panel 1 (le t) and Panel 2 (right).

3T3-L1 cells induced oradipogenesis and thenstained with Oil Red Odemonstrated a signi cantincrease in adipogenesisversus uninduced con-trol cells. TNF , a knowninhibitor o adipogenesis,prevented di erentiation o 3T3-L1 cells.

O D 4 9 0 n m

NoInducers

+ Inducers + Inducers+ TNF

0.5

0.4

0.3

0.2

0.1

0

18

16

14

12

10

M F I ( x 1 0 0 0 )

HepG2 Lysate (ng/well)

8

6

42

01 10 100 1000 10000

ACAA2 LPBE SCHAD TFP

ll

30

25

20

15 M F I ( x 1 0 0 0 )

HepG2 Lysate (ng/well)

10

5

01 10 100 1000 10000

CPT2 DECR1 ETF MCAD MFE2

Key Products

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Transcription FactorsTranscription actors bind speci c DNA sequences andcontrol transcription o adjacent genes. These signalingproteins cooperate with a number o other proteins, suchas coactivators, chromatin remodelers, histone acety-lases, deacetylases, kinases, and methylases, to promoteor block the recruitment o RNA polymerase to speci c

Anti-p53 (pantropic), clone DO-1(Catalogue No. MABE327)

p53 is a ubiquitous transcription actor that is mostwidely known or its unction as a tumor suppressor,through its regulation o cell growth and survival.p53 is activated in response to cell stress such as DNAdamage and induces either cell cycle arrest or apoptosis.It coordinates the expression o multiple genes includingp21, Bax, and Puma. p53 undergoes phosphorylation andacetylation, which may regulate its activity. It may alsobe ubiquitinated via the Akt-MDM2 pathway, resultingin degradation by the 20S proteasome. Mutations in p53are prevalent in many types o cancers, underscoring thetherapeutic relevance o this protein.

Nuclear expression o p53 in A431 cells (A) as well as humancolorectal cancer cells (B) shown by immunocytochemicalstaining using Anti-p53 (pantropic), clone DO-1. Staining wasvisualized with a Donkey Anti-Mouse IgG conjugated to a redfuorescent dye. Actin has been labeled with AlexaFluor 488dye - Phalloidin (Green).

genes. Regulation o transcription actors by upstreammodi ers ensure exact spatial and temporal expressiono genes. Transcription actors are activated sequentiallyand they may also recruit coregulators, coactivators, orcorepressors.

Nuclear SignalingThe nucleus, home to gene expression and regulation, is the endpoint o many signaling pathways,as cells respond to stimuli by altering gene expression patterns. Transcription actors, cell cyclecheckpoint proteins, DNA damage detection and repair mechanisms are all key to nuclear signaling,gene expression and maintaining the integrity o the genome. Furthermore, gene expression isdetermined not only by hereditary in ormation, coded in DNA sequence, but also by epigeneticmarks, or modi cations o DNA and associated proteins that are not necessarily inherited. Epigeneticmechanisms include modi cations to histones, methylation o DNA, remodeling o chromatin,and signaling via noncoding RNA molecules. While the genome remains relatively static, thecomplementary epigenome adapts to environmental infuences and can con er unique characteristicsad phenotypes to di erent cell types, tissues, and organisms. Todays biomedical researchersunderstand that epigenetic marks are as important as DNA sequence in completing the cells complexsignaling network.

A B

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Anti-SMAD2(Catalogue No. 04-1029 )

SMAD2 is one o the TGF receptor-regulated SMADtranscription actors. When SMAD2 is phosphorylatedupon binding o extracellular TGF/activin to receptors,SMAD2 activates transcription o sel -renewal genes,such as Nanog. Interestingly, under di erent cellular

conditions, Wnt signaling cooperates with SMAD2 toactivate di erentiation genes. SMAD2 there ore is acentral switch in signaling pathways that determinecell ate.

Anti-FOXO1(Catalogue No. 05-1075 )

Forkhead box O (FOXO) transcription actors modulatemetabolic unctions. Given the relatively high expressiono FOXO1 in insulin-responsive tissues, this transcription

actor is poised to regulate energy metabolism. Whennutrient and insulin levels are low, FOXO1 promotesexpression o gluconeogenic enzymes. Conversely, in the

ed state, insulin levels rise, stimulating glucose uptakeand inhibiting FOXO1 activation o gluconeogenesis.

Under certain pathophysiologic conditions, includ-ing insulin resistance, negative signaling to FOXO1 iscompromised.

Smad2 is expressed intumors, in which cells areactively undergoing bothsel -renewal and di erentia-tion. Immunohistochemicalstaining o para n-embed-ded human adenocarcinomao uterususing Anti-Smad2(Cat. No.04-1029).

FOXO1 localizes to the nucleio A431 and HeLa cells asdetermined using con ocalfuorescent immunocyto-chemistry with Anti-FOXO1(red, Cat. No.05-1075). Alsoshown are actin (green) andnuclei (blue).
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Documents

Signaling - Antibodies, Proteins, Kit and Assays Product Selection Guide