MILLIPLEX MAP and Cell Signaling Pathways

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1 Data Sheet Product Selection Guide MILLIPLE MAP and Cell Signaling Pathways Your Source for Luminex xmap Technology Growth IGF-R Ins-R PDGF-Rα PDGF-Rβ EGF-R c-met c-kit Cytokines CD3ε TCR CD3ζ slg CD79α Stress TGFβ 1,2,3 Death IRS-1 JAK2 JAK1 Fyn LAT Lck Syk CD79β TGFβR FASL TNFαR GSK3 β-catenin Src PI3K Ras BAD p7 56k Akt Raf FRHD MYPT1 mtor STAT6 STAT5 STAT2 STAT1 Rsk Mek1/2 Erk1/2 Msk1 FAK STAT1 STAT3 Rsk CREB ATF1 Histone 3 ZAP7 Ras MKK3 IKK p38 IκBα NFκB PRAK Hsp27 Smad2/3 Caspase 8 BAD MKK3/6 MKK4/7 p38 JNK Bcl2 Caspase 3 cdc25c cdc2 Aurora 2 ATF2 Jun Smad2/3 PARP Protein Synthesis Apoptosis Transcription Brown = Available from Millipore

2 MILLIPLE MAP and Cell Signaling Pathways Determining 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, MILLIPLE 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, an ever expanding MAPmate portfolio, or PathwayProfiler Service, which features the expertise of our BioMarker Services team. 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 MILLIPLE MAP Cell Signaling Kits MILLIPLE MAP MAPmate Products MILLIPLE MAP Buffer and Detection Kit Cell Lysates Species Cross-reactivity Comparison Data PathwayProfiler Services

3 An example of the power of our MILLIPLE MAP Cell Signaling Portfolio over Western blots is 1 2 demonstrated in the graphs below. Jurkat cells were treated C35 with Isotype C35 Control 9 anti-tcr antibody C35 18 anti-tcrβ 8 CD3ζ 16 CD3ε or an isotype-matched control antibody at 2 µg/ml over a 1-minute period. The MILLIPLE MAP Human T Cell Receptor Kit was used to 5measure the effects of the antibody versus control on the phosphorylation of panel analytes over time. The average MFI and standard 82 CD3ζ 164 CD3ε 71 Isotype 142 Control deviation of 4 replicate wells are shown. Data is representative of 3 replicate experiments. 1 The Westerns shown demonstrate the advantage 4 2 of using C35 MILLIPLE Isotype Control 9 Minutes of Stimulation MAP Cell Signaling 8kits CD3ζ 164 CD3ε and MAPmate products. Isotype 2 Control The Effects of an Anti-TCR 1 on Phosphorylation NT 1 min 2 min 5 min 1 min NT 1 min 2 min 5 min 1 min in C35 Jurkat anti-tcrβ Cells using the Human 25T Cell Receptor 1 2 5Kit 1 (Catalogue No ) Isotype Control NT NT 1 min min 2 min min 5 min min 1 1 min 2 min C35 anti-tcrβ C35 Isotype Control CD3ε Isotype 14 Control C35 C35 Isotype Control CD3ε Minutes of Stimulation NT 1 min 2 min 5 min 1 min C35 anti-tcrβ 1 Minutes of of Stimulation Isotype Control NT NT 1 min min 2 min min 5 min min 1 1 min 5. min C35 anti-tcrβ C35 Isotype Control ZAP7 Isotype 3.5 Control C35 C35 Isotype Control ZAP Minutes of Stimulation Minutes of of Stimulation Average MFI MFI (1x) Average MFI (1x) Average MFI MFI (1x) Average MFI (1x) 1 C35 anti-tcrβ 9 8 Erk Isotype 7 Control Erk Average MFI MFI (1x) Average MFI (1x) C35 anti-tcrβ Isotype Control NT 1 min 2 min 5 min 1 min NT NT 1 min min 2 min min 5 min min 1 1 min min C35 Isotype Control C35 C35 Isotype Control Minutes of Stimulation Minutes of of Stimulation Average MFI (1x) Average MFI (1x) Average MFI (1x) C35 4anti-TCRβ 3 Average MFI (1x) Average MFI (1x) Average MFI (1x) 25 C35 1 anti-tcrβ 25 LCK Isotype Control LCK C35 anti-tcrβ 5. C anti-tcrβ 4. LAT Isotype 3.5 Control C35 2. anti-tcrβ LAT Isotype Control LAT C35 anti-tcrβ Average MFI (1x) Average MFI (1x) Average MFI (1x) C35 Isotype Control Minutes of Stimulation NT 1 min 2 min 5 min 1 min Minutes C35 of Stimulation Isotype Control NT 1 min 2 min 5 min 1 min Minutes of Stimulation NT 1 min 2 min 5 min 1 min Minutes C35 of Stimulation Isotype Control NT 1 min 2 min 5 min 1 min Minutes of Stimulation Isotype Control NT 1 min 2 min 5 min 1 min 5. C35 anti-tcrβ C35 Isotype Control CREB Isotype 3.5 Control NT 1 min 2 min 5 min 1 min 5. C35 2. anti-tcrβ C35 Isotype Control CREB Isotype Control C35 Isotype Control 4.5 Minutes of Stimulation CREB Minutes of Stimulation Average MFI (1x) Average MFI (1x) Average MFI (1x) C35 anti-tcrβ 2 LCK Isotype Control 15 Isotype Control C35 Isotype Control NT 1 min 2 min 5 min 1 min Minutes of Stimulation C35 Isotype Control Minutes of Stimulation NT 1 min 2 min 5 min 1 min C35 Isotype Control C35 Isotype Control Minutes of Stimulation C35 anti-tcrβ Isotype Control Average MFI (1x) Average MFI (1x) Average MFI (1x) Isotype 14 Control C35 anti-tcrβ NT 1 min 2 min 5 min 1 min Minutes C35 of Stimulation Isotype Control NT 1 min 2 min 5 min 1 min C35 8anti-TCRβ 6 Minutes of Stimulation Isotype 4 Control 2 NT 1 min 2 min 5 min 1 min C35 anti-tcrβ C35 Isotype Control 4.5 Minutes of Stimulation 4. ZAP7 Isotype 3.5 Control 3. C35 anti-tcrβ C35 Isotype Control ZAP NT 1 min 2 min 5 min 1 min Isotype Control C35 Isotype Control 4.5 Minutes of Stimulation ZAP Minutes of Stimulation 1..5 NT 1 min 2 min 5 min 1 min C35 anti-tcrβ Minutes of Stimulation Average MFI (1x) Average MFI (1x) Average MFI (1x) Isotype Control 1 C35 anti-tcrβ 9 8 Erk Isotype 7 Control C35 anti-tcrβ Erk Isotype 71 Control Erk Average MFI (1x) Average MFI (1x) Average MFI (1x) NT 1 min 2 min 5 min 1 min NT 1 min 2 min 5 min 1 min C35 Isotype Control C35 Isotype Control NT 1 min 2 min 5 min 1 min C35 Isotype Control NT 1 min 2 min 5 min 1 min C35 Isotype Control Minutes C35 of Stimulation Isotype Control Minutes of Stimulation Minutes of Stimulation

4 MILLIPLE MAP Cell Signaling Kits and MAPmate Panels 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 AVAILABLE ANALYTES/ THERAPEUTIC AREAS* Oncology Inflammation Metabolic Syndrome Autoimmune Stem Cells Apoptosis CNS activated by a ligand interacts directly with Akt/PKB 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 ATF2 BAD Caspase 3 CREB EGFR Erb-B2/HER2 Erk/MAPK 1/2 HSP27 IGF-1R IκBα IRS1 JNK c-jun c-kit feedback, signal amplification, and interactions Lck between more than one signal and among several c-met/hgfr 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. p38 p53 p7 S6 Kinase PARP PDGFRα PDGFRβ Rsk1 STAT1 STAT3 STAT5A/B STAT6 ZAP-7 *Based on citation frequency in PubMed.

5 MILLIPLE MAP Cell Signaling Kits Analytes featured in Millipore s MILLIPLE MAP Cell Signaling Kits have been analytically validated together. Each kit includes all the reagents necessary to run an assay on µL samples in duplicate all in one kit, with one catalogue number: Beads (capture antibodies) Biotin (detection antibodies) Lysis buffer Assay buffer(s) Stimulated and unstimulated cell lysates (positive and negative controls) Streptavidin-Phycoerythrin Opaque multi-screen plate and cover Mixing vials User-friendly protocol Environmentally-friendly packaging Human T Cell Receptor 7-Plex (Catalogue No ) MILLIPLE m a p T Cell Receptor Kit is a 7-plex assay that examines seven phosphorylated proteins involved in immune response, tumor development and inflammation. Analyte CD3e CREB Erk/MAPK 1/2 LAT Phosphorylated (pan Tyr) (Ser133) (Thr185/Tyr187) (pan Tyr) Analyte Lck Syk ZAP-7 Phosphorylated (pan Tyr) (pan Tyr) (pan Tyr) Multi-Pathway 8-Plex (Catalogue No ) MILLIPLE m a p Multi-Pathway Kit is an 8-plex assay that examines eight phosphorylated proteins that span several signal transduction pathways including MAPK, inflammation and apoptosis. Analyte CREB Erk/MAPK 1/2 IkBa JNK Phosphorylated (Ser133) (Thr185/Tyr187) (Ser32) (Thr183/Tyr185) Analyte p38 p7 S6 Kinase STAT3 STAT5A/B Phosphorylated (Thr18/Tyr182) (Thr412) (Ser727) (Tyr694/699) Human Apoptosis 3-Plex (Catalogue No ) Analytes Active Caspase 3 Active Cleaved PARP Apoptosis plays a fundamental role in many normal biological processes, including development, homeostasis and DNA repair. MILLIPLE m a p Human Apoptosis Kit is currently a 3-plex assay that examines active caspase-3 and cleaved PARP, proteins involved in immune response, tumor development and inflammation. GAPDH is provided as a control for protein loading. This panel will be expanded to a 7-plex in 21. Additional MAPmate products are also available for the study of apoptosis. GAPDH Human MAP Kinase 1-Plex (Catalogue No ) The complex MAP Kinase pathway connects intracellular responses to the binding of growth factors via cell surface receptors. The MILLIPLE m a p MAP Kinase Kit is a 1-plex assay that examines ten phosphorylated proteins involved in normal mitosis and tumor development. Analyte ATF2 ERK/MAP Kinase 1/2 HSP27 JNK cjun Phosphorylated (Thr69/71) (Thr185/Tyr187) (Ser78) (Thr183/Tyr185) (Ser73) Analyte MEK1 Msk1 p38 p53 STAT1 Phosphorylated (Ser221) (Ser212) (Thr18/Tyr182) (Ser15) (Tyr77) Human Src Family Kinase Family (SFK) 8-Plex (Catalogue No ) Analytes Src family of proto-oncogenic kinases (SFK) is the largest nonreceptor protein tyrosine kinase family consisting of: Src, Yes, Fyn, Fgr, Lck, Hck, Blk, and Lyn. Expressed either ubiquitously or predominately in hematopoietic immune-competent cells, members of the SFK play a crucial role in embryonic development and cell growth, regulating such functions as proliferation, adhesion, differentiation, and survival that are common to both cellular processes. Consequently, these signaling proteins are often fundamentally involved in the oncogenesis of tumors such as non-small cell lung cancer (NSCL), squamous cell head and neck and pancreatic cancers. Blk Fgr Fyn Hck Lck Lyn Src Yes

6 MILLIPLE MAP MAPmate Portfolio: Building Custom Intracellular Assays If you prefer to design a custom assay, Millipore has simplified this process for you, making ordering the necessary kit components easier so that you are assured you have everything you need to do your best work. Step 1. Plan your assay Choose the analytes you want to plex together and determine their compatibility. Keep in mind that total and phosphorylated analytes cannot be plexed together. Using the following buffer table, analytes that use the same assay buffer can be included in your custom assay. This enables you to concentrate on proteins belonging to a specific signal transduction pathway or to look at downstream targets that have a role in multiple pathways. Total MAPmate Portfolio Catalogue No. Description Bead Region Lysis Buffer / Assay Buffer Total Akt/PKB, Assay Buffer 1 38 l Total Akt/PKB, Assay Buffer l Total ATF2 39 l l Total Bad 59 l l Total c-kit 8 l l Total c-met/hgfr 46 l l Total CREB 37 l l Total EGF Receptor 34 l l Total ErbB2/Her2 29 l l Total ERK 1/2 9 6 l Total Hsp27 5 l Total IGF1R 48 l l Total IkBa 33 l Total IRS1 36 l l Total JNK 16 6 l Total Lck 2 l Total MEK1 45 l l Total MKK l Total MSK l Total p21 28 l Total p l Total p l Total p7 S6 Kinase 19 l l Total STAT1 24 l Total STAT2 67 l l Total STAT3 14 l Total Human GAPDH 15 6 l For the most updated version of this table, visit Then click the MAPmate Buffer Table link. l Recommended Acceptable 6 Not Recommended

7 Phosphorylated MAPmate Portfolio Catalogue No. Description Bead Region Lysis Buffer / Assay Buffer Active Caspase 3 6 l l Cleaved PARP (Asp214) 6 l l Phospho Akt/PKB (Ser473), Assay Buffer 1 38 l Phospho Akt/PKB (Thr38), Assay Buffer 1 38 l Phospho Akt/PKB (Ser473), Assay Buffer l Phospho Akt/PKB (Thr38), Assay Buffer l Phospho ATF2 (Thr69/71) 39 l l Phospho Bad (Ser112) 59 l Phospho c-jun (Ser73) 52 l Phospho c-kit (Tyr) 8 l l Phospho c-met/hgfr (Tyr) 46 l l Phospho CREB (Ser133) 37 l l Phospho EGF Rec (Tyr) 34 l l Phospho ErbB2/Her2 (Tyr) 29 l Phospho ERK 1/2 (Thr185/Tyr187) 9 6 l Phospho Histone H2A. (Ser139) 43 l Phospho Hsp27 (Ser78) 5 l l Phospho IGF1R (Tyr) 48 l l Phospho IkBa (Ser32) 33 l Phospho IRS1 (Tyr) 36 l l Phospho JNK/SAPK1 (Thr183/Tyr185) 16 6 l Phospho Lck (Tyr) 2 l Phospho MEK1 (Ser222) 45 l Phospho MKK6 (Ser27/Thr211) 66 l Phospho MSK1 (Ser212) 75 6 l Phospho p38/sapk (Thr18/Tyr182) 54 6 l Phospho p53 (Ser15) 53 l Phospho p53 (Ser46) 53 l Phospho p7 S6 Kinase (Thr412) 19 6 l Phospho PDGF Receptorb (Tyr) 32 l Phospho PDGF Receptora (Tyr) 21 l Phospho Rsk1 (Ser38) 42 l Phospho STAT1 (Tyr71) 24 l Phospho STAT2 (Tyr69) 67 l l Phospho STAT3 (Ser727) 14 l Phospho STAT3 (Tyr75) 14 l Phospho STAT5A/B (Tyr694/Tyr699) 35 l Phospho STAT6 (Tyr641) 1 l l Phospho ZAP-7 (Tyr) 51 l For the most updated version of this table, visit Then click the MAPmate Buffer Table link. l Recommended Acceptable 6 Not Recommended

8 Step 2. Order the MILLIPLE MAP Cell Signaling Buffer and Detection Kit (Catalogue No ) With the exception of MAPmates, this buffer kit contains everything you need to run your assay. The kit includes: MILLIPLE MAP Lysis Buffer MILLIPLE MAP Assay Buffer 1 MILLIPLE MAP Assay Buffer 2 MILLIPLE MAP Amplification Buffer Streptavidin-Phycoerythrin Negative / positive control (Phosphorylated / Total) Opaque 96-well Filter Plate and cover Empty mixing vials This buffer and detection kit is required for MAPmate analysis of samples. Step 3. Order the MAPmate products you have selected A MAPmate consists of: Capture (bead) Detection (biotin) Positive Control (stimulated cell lysate) for phosphorylated analytes The positive control can be used to qualify assay performance or, with serial dilutions, to develop a standard curve. ordering is as easy as The MILLIPLE MAP buffer kit and MAPmate products will provide you with all the components and reagents you need to simultaneously detect the multiple analytes you have chosen to study.

9 MILLIPLE MAP GAPDH Unique to Millipore The Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) gene is constitutively and stably expressed in almost all tissues and cells, and is considered to be a housekeeping gene. Millipore has developed a human GAPDH MAPmate to aid you in your cellular experiments, using it as a loading control and for protein normalization in your multiplex assays. Ave MFI 8, 6, 4, 2, GAPDH Human Lysate Titration using Assay Buffer 2 HeLa HEK293 A431 A549 Kasumi MCF7 Jurkat Total GAPDH HeLa Concentration (µg/ml) GAPDH cellular concentrations can vary significantly depending on the cell line. An initial sample titration similar to that shown in the representative data above is recommended for any previously untested cell line. Cell Lysates: Positive and Negative Controls Our stimulated and unstimulated cell lysates were designed to be used as controls with MILLIPLE MAP Cell Signaling Kits and MAPmate products. These lysates are also recommended for use in Western blotting and immunoprecipitation. Available Lysates Catalogue No. Control Sample Catalogue No. Control Sample HeLa: unstim MCF-7: IGF Jurkat: unstim Daudi: IL Jurkat: anisomycin A549: camptothecin Jurkat: H 2 O T3: anisomycin A431: EGF T3: unstim HeLa: HS/Ars HeLa: HGF HeLa: TNFa Ramos: Pervanadate NIH3T3: PDGF HL-6: Pervanadate Kasumi-3: SCF HeLa: IFNa HEK293: LiCl

10 Cell Lysates: Positive and Negative Controls (continued) The graphs below show representative data obtained when using Millipore s cell lysates in each of the below assays. In each case stimulated and unstimulated lysates were resuspended in ultrapure water. MILLIPLE MAP: A serial dilution of a rehydrated lysate was analyzed using an appropriate MAPmate. The Median Fluorescence Intensity (MFI) was measured with the Luminex Instrument. MILLIPLE MAP Detection Western Blot: Aliquots of lysate were analyzed using the appropriate MAPmate of Cleaved PARP in Jurkat Cells 4,5 biotin labeled detection antibody and imaged using Streptavidin-HRP and chemiluminescence. IP/Western: Aliquots of lysates were mixed with the appropriate MAPmate capture antibody beads to immunoprecipitate the protein. The immunoprecipitated proteins were separated on SDS-PAGE, transferred to nitrocellulose, and probed 5 with the same MAPmate biotin-labeled detection antibody. The proteins were imaged using Streptavidin-HRP and chemiluminescence. MFI 4, 3,5 3, 2,5 2, 1,5 1, Jurkat: unstimulated Jurkat: anisomycin mg of Cell Lysate per Well MFI 4,5 4, 3,5 3, 2,5 2, 1,5 1, 5 MILLIPLE MAP Detection of Cleaved PARP in Jurkat Cells Jurkat: unstimulated Jurkat: anisomycin Western Blot Cleaved PARP + anisomycin IP/Western Cleaved PARP + anisomycin mg of Cell Lysate per Well Western Blot Cleaved PARP IP/Western Cleaved PARP + anisomycin + anisomycin

11 MILLIPLE MAP Intracellular Assays: Species Cross-Reactivity While most of the capture and detection antibodies are anti-human, many are cross-reactive with other species. In most cases these analytes were identified by examining amino acid sequence to determine homology. The table below illustrates species cross-reactivity for each MAPmate product. Total MAPmate Portfolio Catalogue No. Description Species Reactivity Human Mouse Rat Total Akt/PKB, buffer 1 l l l Total Akt/PKB, buffer 2 l l l Total ATF2 l l Total Bad l l Total c-kit l Total CREB l l l Total EGF Receptor l Total ErbB2/Her2 l Total Erk 1/2 l l l Total Hsp27 l Total IGF1R l Total IkBa l Total IRS1 l l l Total JNK l l Total Lck l l Total MEK1 l l Total c-met/hgfr l Total MKK6 l l l Total MSK1 l Total p21 l Total p38 l l l Total p53 l Total p7 S6 Kinase l l l Total STAT1 l l Total STAT2 l Total STAT3 l l l Total Human GAPDH l For the most updated version of this table, visit Then click the Species Cross-Reactivity Table link.

12 Phosphorylated MAPmate Portfolio Catalogue No. Description Species Reactivity Human Mouse Rat Phospho-Akt/PKB (Ser473), buffer 1 l l l Phospho-Akt/PKB (Ser473), buffer 2 l l l Phospho-Akt/PKB (Thr38), buffer 1 l l l Phospho-Akt/PKB (Thr38), buffer 2 l l l Phospho-ATF2 (Thr69/71) l l Phospho-Bad l l Active Caspase 3 l l Phospho-CREB (Ser133) l l l Phospho-EGFR (Tyr) l Phospho-ErbB2/Her2 (Tyr) l Phospho-Erk 1/2 (Thr185/Tyr187) l l l Phospho-Histone H2A. l l Phospho-Hsp27 (Ser78) l Phospho-IGF1R (Tyr) l Phospho-IkBa (Ser32) l Phospho-IRS1 (Tyr) l l l Phospho-JNK/SAPK1 (Thr183/Tyr185) l l Phospho-c-Jun (Ser73) l l l Phospho-c-Kit (Tyr) l Phospho-Lck (Tyr) l l l Phospho-MEK1 (Ser222) l l l Phospho-c-Met/HGFR (Tyr) l Phospho-MKK6 (Ser27/Thr211) l l Phospho-MSK1 (Ser212) l Phospho-p38/SAPK (Thr18/Tyr182) l l l Phospho-p53 (Ser15) l Phospho-p53 (Ser46) l Phospho-p7 S6 Kinase (Thr412) l l l Cleaved PARP (Asp214) l Phospho-PDGF Receptor a l l Phospho-PDGF Receptor b l Phospho-Rsk1 (Ser38) l l l Phospho-STAT1 (Tyr71) l l Phospho-STAT2 (Tyr69) l Phospho-STAT3 (Ser727) l l l Phospho-STAT3 (Tyr75) l l l Phospho-STAT5A (Tyr694) l l l Phospho-STAT6 (Tyr641) l Phospho-ZAP7 (Tyr) l For the most updated version of this table, visit Then click the MAPmate Buffer Table link.

13 MILLIPLE MAP Comparative Data Performance characteristics of MILLIPLE MAP Cell Signaling MAPmate products were benchmarked against analytes selected from two competitors. A total of five phosphoprotein analytes (Table 1) were selected based on availability from Millipore and competitor companies, as well as maximal signaling pathway overlap. Advantages of MILLIPLE MAP Cell Signaling Portfolio: Simpler, Easier Multiplexing Protocol and Assay Design: o Very straightforward and user-friendly o No additional sample treatment for some MAPmates o One protocol design for all MAPmate products and kits Kit Format: o All kits designed with the same format for multiplexing Comparison of Assays with the least optimization using Normalized Lysates 1 Buffers Required: o Only two buffers 8 required Dilution and Wash buffers o Overnight incubation 4 at 4 C 2 6 Incubation Time and Temperature: o Shorter incubation time at room temperature possible Standards and Controls: o Lyophilized negative control (unstimulated lysate) included in MILLIPLE MAP Buffer and Detection kit pegfr perk1/2 p-p7 S6K pjnk/sapk1 p-p38 Millipore Competitor A Competitor B o Lyophilized positive control (stimulated lysate) included with phospho-mapmates Target pegfr pjnk/sapk1 perk1/2 p-p38 p-p7 S6K Table 1. A pegfr perk1/2 p-p7 S6K Lysate Tested A431/EGF HeLa/HS-Ars A431/EGF HeLa/HS-Ars A431/EGF Comparison of Assays using Normalized Lysates pjnk/sapk1 p-p38 Millipore Competitor A Competitor B B Comparison of Assays: Fold Induction 25 1 Comp F pegfr perk1/2 p-p7 S6K pjnk/sapk1 5 p-p Millipore Competitor A Competitor B To determine fold induction the stimulated average MFI was divided by the unstimulated average MFI. Figure A and B. Manufacturer s instructions for both cell lysis, sample preparation and for protocol assay procedures were followed for each of the cell signaling kits (Catalogue Nos , 46-62, , 46-61, and ). The results below showing the fold induction values, as well as fold induction with normalized lysates, demonstrate the superior product performance of the MILLIPLE MAP Cell Signaling product line. pegfr Millipore perk1/2 Competitor A p-p7 S To determine fold induction the stimulated average MFI was d

14 Millipore s BioMarker Services Presents MILLIPLE MAP PathwayProfiler Service PathwayProfiler Service is a convenient, time-saving service for the simultaneous investigation of customer-defined signal transduction events in a native cellular environment. Millipore s world-leading expertise in phospho-specific antibodies and the power of multiplex assays offer you an unimpeded view into cellular response. Use PathwayProfiler Service to screen protein modifications of off-target signaling pathways or to develop a more complete biological understanding of in vivo compound efficacy in a wide variety of cellular environments. In addition, the capability to combine our PathwayProfiler and KinaseProfiler services provides our customers with the ability to determine relationships between specific kinases and complex signal transduction pathways. Free yourself from the headache of Western blotting, and realize all the benefits of cell signaling multiplex assays. Investigate multiple kinase signaling cascades in the same sample well, minimizing variations of experimental conditions in a robust and economical methodology. Simply prepare your cell lysates, choose your targets (total and phosphorylated) and leave the rest to Millipore s BioMarker Services Team. Samples and controls are handled and analyzed by highly trained technicians, following strict protocols with precision and reliability. Customers receive a report with protein and phosphoprotein levels for each sample provided. The report also includes raw data, normalized data and complete quality control information. PathwayProfiler Service is: o A cell-based assay o Multiplexed phospho-specific readout o Drug testing in physiological conditions o Precise and reliable results o User customizable With an ever expanding panel, PathwayProfiler Service enables you to screen your cell lysates against either all analytes or only those of interest to you. Applications include: o Target identification o Target validation o Compound selectivity o Monitoring phosphorylation status of kinases and substrates Measuring cell-based signaling cascades provides a confident assessment of drug effects. With Millipore s PathwayProfiler Service, you can: Test drug effectiveness at physiological ATP and substrate concentrations Examine drug effects on multiple targets in a single sample Get precise and reliable results no more Western blotting Test multiple cell types or cells derived from various disease states

15 Valuable Results with Millipore s BioMarker Services PathwayProfiler Service is Millipore s newest service part of a powerful suite of services to quickly and conveniently provide you with valuable research data. Using PathwayProfiler Services is simple: contact Millipore, and prepare your cell lysate samples with the lysate buffer we provide. Then, freeze and ship your sample to us. Our technical service team is here to support you through each step of the process, ensuring your complete satisfaction and timely results. Customer/Researcher Modify Compound/ Cell Type Millipore Receive and Analyze Data BioMarker Services expert staff runs customized assays and sends data Send Cell Lysate and Order Form to Millipore PathwayProfiler Service Treat Cells with Lead Compound and Stimulant Lyse Cells with Millipore Lysis Buffer (43-2) Freeze Cell Lysate at -8 C Customization: Choose Target Proteins (Total and/or Phospho) PathwayProfiler Service is the MILLIPLE service solution to your running numerous Western blots. Advantages include: Small Sample Volumes Consistent quality control Expertise in High Quality Sample Analyses Guaranteed Reproducibility Prompt & Flexible Reporting Leader in Multiplex Services intracellular and extracellular Long and Proven History with Clients Two- to four-week turnaround time, depending on project For a complete list of available total and phosphorylated analytes, see

16 Luminex xmap Technology With xmap technology, you will benefit from the ability to simultaneously quantitate up to 1 analytes in a single sample, using as little as 25 µl or less. Speed/High-Throughput: Simultaneously measure the concentration of a large number of analytes, allowing you to do more faster, gaining early and comprehensive data so critical to your work. Accuracy: xmap technology generates real-time analysis and accurate quantification of biological interactions. Reproducibility: The high-volume production of xmap beads allows assay standardization that solid-phased flat arrays cannot provide. xmap technology process flow Bead #56, TNF-α Bead #29, IFN-γ Bead #12, IL-6 1 Luminex uses proprietary techniques to internally color-code microspheres with two fluorescent dyes. Through precise concentrations of these dyes, 1 distinctly colored bead sets can be created, each of which is coated with a specific capture antibody. 2 After an analyte from a test sample is captured by the bead, a biotinylated detection antibody is introduced. The reaction mixture is then incubated with Streptavidin PE conjugate, the reporter molecule, to complete the reaction on the surface of each microsphere. 3 The microspheres are allowed to pass rapidly through a laser, which excites the internal dyes marking the microsphere set. A second laser excites PE, the fluorescent dye on the reporter molecule. COMPARISON OF THE xmap & WESTERN BLOT TECHNOLOGIES Side-by-side comparison of Multiplexing vs. Western Blot Traditional Method Multiplexing Western 1 Protein/probe Western on a Bead Total Proteins Phosphorylated Proteins

17 MILLIPLE MAP: Everything You Need All In One Place Products Higher Quality Industry-leading QC, QA and Six Sigma Principles. More Efficient Analytically validated kits for consistency and reproducibility. All-in-One Kit A single kit with a single catalogue number. More Flexible Select only the analytes needed or choose a pre-mixed kit. Continuity of Knowledge Is the Key Millipore owns every step of the process, MILLIPLE MAP from R&D to manufacturing. More Choices The broadest range of multiplex analytes for the Luminex xmap platform. Instruments Speed/High-Throughput Simultaneously measure the concentration of a large number MILLIPLE Analyzer of different analytes, allowing you to do more faster, gaining early and comprehensive data so critical to your work. Accuracy xmap technology generates real-time analysis and accurate quantification of the biological interactions. Reproducibility High-volume production of xmap microspheres allows assay standardization that solid-phased flat arrays cannot provide. Low Sample Volume With minimal hands-on time, you can screen more than 4 analytes in a single sample using as little as 25 µl or less. Software The Luminex xponent software helps you manage and track all of your multiplex assays by combining ease of use and flexibility to perform both protein and nucleic acid MILLIPLE Analyst testing on one system. It s also compatible with the upcoming magnetic bead (MagPlex beads) assays, and will be moving forward with compatible future instrument platforms. NEW! Millipore now offers MILLIPLE Analyst software, developed exclusively for Millipore by VigeneTech. This data analysis package helps you do your best work with increased speed and efficiency. MILLIPLE Analyst software provides a whole new level of data analysis by allowing you to import large data sets and benefit from the ability to calculate your results in seconds. The user-friendly plate setup feature lets you work with speed and efficiency while the unique algorithms on even the most challenging data sets give you the performance you need for multiplexing analysis. Services Millipore s BioMarker Services has been performing immunoassays for over 25 years. Assay services are available for RIAs, ELISAs, IRMAs and multiplex assays, using both Millipore kits as well as any commercially available assays. Quality control, reproducible results, and confidentiality are of paramount importance to BioPharma Services our BioMarker Services team. Millipore s Regulatory Compliant Laboratory is uniquely positioned to provide bioanalytical services to support your preclinical and clinical studies. For a complete and up-to-date list of MILLIPLE MAP analytes, visit

18 ORDERING INFORMATION Description Quantity Catalogue No. Multi-Pathway 8-Plex 96-well plate Human T Cell Receptor 7-Plex 96-well plate Human Apoptosis 3-Plex 96-well plate Human MAP Kinase 9-Plex 96-well plate Cell Signaling Buffer and Detection Kit 96-well plate MILLIPLE MAP MAPmate Products 96-well plate Description Phospho Akt/PKB (Ser473), Assay Buffer 1 Catalogue No Phospho ERK 1/2 (Thr185/Tyr187) Phospho EGF Rec (Tyr) Active Caspase Total Akt/PKB, Assay Buffer Total EGF Receptor Phospho Hsp27 (Ser78) Total Hsp Total ERK 1/ Phospho p38/sapk (Thr18/Tyr182) Phospho Rsk1 (Ser38) Total p Phospho JNK/SAPK1 (Thr183/ Tyr185) Phospho PDGF Receptora (Tyr) Phospho PDGF Receptorb (Tyr) Phospho Lck (Tyr) Total Lck Total JNK Phospho c-kit (Tyr) Total c-kit Total p Phospho c-jun (Ser73) Phospho STAT3 (Tyr75) Phospho STAT3 (Ser727) Total STAT Phospho IRS1 (Tyr) Total IRS Phospho p7 S6 Kinase (Thr412) Total p7 S6 Kinase Phospho CREB (Ser133) Total CREB Phospho STAT6 (Tyr641) Total STAT Phospho STAT2 (Tyr69) Description Phospho STAT5A/B (Tyr694/ Tyr699) Catalogue No Phospho IkBa (Ser32) Total IkBa Phospho Akt/PKB (Thr38), Assay Buffer 1 Total IGF1R Phospho IGF1R (Tyr) Total ErbB2/Her Phospho ErbB2/Her2 (Tyr) Total c-met/hgfr Phospho c-met/hgfr (Tyr) Total STAT Phospho STAT1 (Tyr71) Cleaved PARP (Asp214) Total ATF Phospho ATF2 (Thr69/71) Phospho ZAP-7 (Tyr) Total p Phospho p53 (Ser15) Phospho p53 (Ser46) Total Human GAPDH Total MEK Phospho MEK1 (Ser222) Total MKK Phospho MKK6 (Ser27/Thr211) Total MSK Phospho MSK1 (Ser212) Total Akt/PKB, Assay Buffer Phospho Akt/PKB (Thr38), Assay Buffer 2 Phospho Akt/PKB (Ser473), Assay Buffer 2 Phospho Histone H2A. (Ser139) Phospho Bad (Ser112) Total Bad

19 MILLIPLE MAP Cell Lysates 96-well plate Description Catalogue No. HeLa: unstimulated Jurkat: unstimulated Jurkat: anisomycin Jurkat: H 2 O A431: EGF HeLa: HS/Ars HeLa: TNFa NIH3T3: PDGF Kasumi-3: SCF Description Catalogue No. HEK293: LiCl MCF-7: IGF Daudi: IL A549: camptothecin T3: anisomycin Jurkat: paclitaxel T3: unstimulated HeLa: HGF Other Related Millipore Products o MILLIPLE MAP Cytokine/Chemokine Multiplex Panels: human, high sensitivity human, non-human primate, mouse, rat, canine o MILLIPLE Analyst o BioMarker Services o KinaseProfiler and Kinase Products o GPCRProfiler and GPCR Products o Cell Signaling Antibodies Millipore, Advancing Life Science Together, MILLIPLE, and GPCRProfiler are registered trademarks of Millipore Corporation. EpiQuant, KinaseProfiler, PathwayProfiler, MAPmate, and the M mark are trademarks of Millipore Corporation. Luminex, xponent, and xmap are registered trademarks of Luminex Corporation. Luminex 2 and MagPlex is a trademark of Luminex Corporation. VigeneTech is a registered trademark of VigeneTech, Inc. Lit. No. PB2723EN, Rev. A 9LS11 Printed in U.S.A. 12/9 LS-SBU Millipore Corporation, Billerica, MA 1821 U.S.A. All rights reserved. VigeneTech

20 Targets contained in Panel 1 are found in many GeneGo Inc. pathways See Millipore s Signaling Pathways Learning Center webpage at: Some examples are shown below. Targets such EGFR and Shc may be present in multiple pathways. Target Apoptosis/Survival Development Pathways Immune Response Chemotaxis EPO Activation Cell Adhesion Cytoskeleton Remodeling Regulation of Lipid Metabolism G-Protein Signaling EGFR / ErbB Development Signal Transduction Transcription Translation Transport Cbl (Y7/731/774) Crk (Y221/239) CUB domain containing protein 1 (Y734) EGFR (Y111/1125) Ezrin (Y477) FAK (Y397/47) FAK (Y861) FRS2 (Y436) Fyb (Y697/651) Gab1 (Y285/37/317) Gab2 (Y266) Gab2 (Y584) GHR (Y566) HER2 (Y123) IL-15R (Y227) IRS1 (Y612) ITK (Y18) JAK3 (Y785) Jnk 1/2/3 (Y185/223) LAT (Y255) HDFR (MET) (Y13) p38 MAPKa (Y181) PDK1 (Y373/376) PI3K p1 (Y485) Plakoglobin (g-catenin) (Y55) PLC1g (Y1253) PLC2g (Y1197/1217) Raf-1 (Y34/341) RapGEF1 (GRF2) (Y54) SCF38 (Y2) SH2 domain containing protein 3A (Y231) SH2 domain containing protein 3A (Y95) Shc (Y349/35) SPTAN1 (Y1176) STAT3 (Y75) Tec (Y26) TfR (Y2) VEGFR1 (Y1169) WASP (Y291) ZAP7 (Y126)

21 Natural Sample Response Prepared cell lysates were assayed using the MPEQ-1K kit: MPEQ-LY1- EpiQuant Jurkat: Unstimulated Lysate MPEQ-LY2- EpiQuant A431: Unstimulated Lysate MPEQ-LY3- EpiQuant Jurkat: H 2 O 2 Stimulated Lysate MPEQ-LY4- EpiQuant A431: H 2 O 2 Stimulated Lysate MPEQ-LY5- EpiQuant A431: EGF Stimulated Lysate Results are tabulated below. Values are the average of triplicate measurements. Target Concentration in Control Lysates (pm) Target MPEQ-LY1 MPEQ-LY2 MPEQ-LY3 MPEQ-LY4 MPEQ-LY5 Cbl (Tyr7/731/774) Crk (Tyr221/239) <.42 < CUB domain containing protein 1 (Tyr734) EGFR (Tyr111/1125).8 < Ezrin (Tyr477) <.13 < FAK (Tyr397/47) < FAK (Tyr861) FRS2 (Tyr436) FTyrb (Tyr697/651 (iso1/2)) 5.9 < Gab1 (Tyr285/37/317) Gab2 (Tyr266) Gab2 (Tyr584) Growth hormone receptor (Tyr566) HER2 (Tyr123) HGFR (MET) (Tyr13) <.4 <.4 < Interleukin-15 receptor a chain (Tyr227) IRS1 (Tyr612) ITK (Tyr18) JAK3 (Tyr785) JNK1/2/3 (Tyr185/185/223).1 <.4.4 <.4.1 LAT (Tyr255) p38/mapka (Tyr181).3 < PDK1 (Tyr373/376) <.1 < <.1 Pl3Kp11d (Tyr485) < Plakoglobin (g catenin) (Tyr548) PLC1g (Tyr1253) PLC2g (Tyr1197/1217) Raf 1 (Tyr34/341) RapGEF1 (GRF2) (Tyr54 (522 in iso1)) 1.6 < SCF38 (Tyr2) SH2 domain containing protein 3A (Tyr231) SH2 domain containing protein 3A (Tyr95) Shc (Tyr349/35) SPTAN1 (Tyr1176) STAT3 (Tyr75) <.27 <.27 < <.27 Tec (Tyr26) 26.3 < TfR (Tyr2) VEGFR1 (Tyr1169) WASP (Tyr291) ZAP7 (Tyr126)

22 Biological Validation for MILLIPLE MAP EpiQuant Analytes All MILLIPLE m a p EpiQuant analytes have been validated for both biological relevance and specificity. Phosphotyrosine Activation in Unstimulated and Stimulated A431 Cells Non EGF H 2 2 Concentration (pm) WASP (Y291) TFR (Y2) TEC (Y26) SPTAN1 SHC (Y349) SDCP3A (Y95) SDCP3A (Y231) SCF38 (Y2) RAF1 (Y34) PLC2γ (Y1197) PLC1γ (Y1253) PLAK (Y548) PDK1 (Y373) p38 (Y181) MET (Y13) JNK (Y185) JAK3 (Y785) ITK (Y18) IRS1 (Y612) IL15Ra (Y227) HER2 (Y123) GHR (Y566) FAK (Y861) FAK (Y397) EZRIN (Y477) EGFR (Y111) CRK (Y221) CDCP1 (Y734) CBL (Y7) A431 cells were treated EGF or H 2 O 2 or vehicle. Following lysis and digestion, EpiQuant beads (a 29-plex) were added to samples containing the target proteins shown. Samples were analyzed on a Luminex 2 reader. Data shown is the mean pm value from 4 observations. Increased phosphorylation was noted for EGFR, Her2, IL-15R, Shc and Tec compared to vehicle treated cells.

23 Dynamic Range:.32 pm - 1, pm Specificity: There is no significant cross-reactivity observed between the antibodies and/or non-specific analytes in this panel. Precision: Intra-Assay: 1.8% 17.5% Inter-Assay: 5.6% 19.6% Accuracy: 81.2% 119.6% Sensitivity: Phosphotyrosine Cell Signaling Panel 1 MinDC <.32 pm MinDC = pm MinDC = pm MinDC > 3.1 pm Cbl (py7/731/774) Crk (py221/239) CUB domain containing protein 1 (py734) Fyb (py697/651 (iso1/2)) EGFR (py111/1125) FAK (py861) FRS2 (py436) LAT (py255) Ezrin (py477) Gab2 (py266) Gab1 (py285/37/317) Plakoglobin (g catenin) (py548) FAK (py397/47) Gab2 (py584) HER2 (py123) SCF38 (py2) JNK1/2/3 (py185/185/223) Growth hormone receptor (py566) IL-15 receptor a chain (py227) SH2 domain containing protein 3A (py95) p38mapka (py181) HGFR (met) (py13) JAK3 (py785) Tec (py26) PDK1 (py373/376) IRS1 (py612) RapGEF1 (GRF2) (py54 (522 in iso1)) VEGFR1 (py1169) Pl3Kp11d (py485) ITK (py18) TfR (py2) WASP (py291) PLC1g (py1253) Shc (py349/35) PLC2g (py1197/1217) SPTAN1 (py1176) Raf 1 (py34/341) STAT3 (py75) SH2 domain containing protein 3A (py231) ZAP7 (py126)

24 Analytes in MILLIPLE m a p EpiQuant Phosphotyrosine Cell Signaling Panel 1 Cbl (Tyr7/731/774) HGFR (Tyr13) Rap GEF1/GRF2 (Tyr54) Crk (Tyr221/239 IL-15Ra chain (Tyr227) SCF38 (Tyr2) CUB domain containing protein 1 (Tyr734) IRS1 (Tyr612) SH2 domain containing protein 3A (Tyr231) EGFR (Tyr111/1125) ITK (Tyr18) SH2 domain containing protein 3A (Tyr95) Ezrin (Tyr477) JAK3 (Tyr785) Shc (Tyr349/35) FAK (Tyr397/47 JNK 1/2/3 (Tyr185/185/223) SPTAN1 (Tyr1176) FAK (Tyr861) LAT (Tyr255) STAT3 (Tyr75) FRS2 (Tyr436) p38a/mapk2a (Tyr181) Transferrin Receptor (Tyr2) Fyb (Tyr97/651) PDK1 (Tyr373/376) Tyrosine protein kinase Tec (Tyr26) Gab1 (Tyr285/37/317) PI3K p11d (Tyr485) VEGFR1 (Tyr1169) Gab2 (Tyr266) Plakoglobin/g-catenin (Tyr548) WASP (Tyr291) Gab2 (Tyr584) PLC1g (Tyr1253) ZAP7 (Tyr126) Growth hormone receptor (Tyr566) PLC2g (Tyr1197/1217) HER2 (Tyr123) Raf-1 (Tyr34/341) Assay Characteristics 1 Peptide Standard Curves Peptide Standard Curves p38mapka (py181) JNK1/2/3 (py185/185/223) 1 Shc (py349/35) EGFR (py111/1125) PDK1 (py373/376) PLC1γ (py1253) Median Fluorescence Intensity 1 1 PLC2γ (py1197/1217) Cbl (py7/731/774) IRS1 (py612) FAK (py861) SH2 domain containing protein 3A (py231) Interleukin-15 receptor a chain (py227) Growth hormone receptor (py566) Tec (py26) WASP (py291) FAK (py397/47) HER2 (py123) Ezrin (py477) Plakoglobin (g-catenin) (py548) JAK3 (py785) Crk (py221/239) CUB domain containing protein 1 (py734) SH2 domain containing protein 3A (py95) Raf 1 (py34/341) SCF38 (py2) ITK (py18) HGFR (met) (py13) TfR (py2) SPTAN1 (py1176) ZAP7 (py126) Pl3Kp11d (py485) STAT3 (py75) Gab1 (py285/37/317) LAT (py255) Fyb (py697/651 (iso1/2) Gab2 (py266) Gab2 (py584) RapGEF1 (GRF2) (py54 (522 in iso1)) VEGFR1 (py1169) FRS2 (py436) [peptide](pm) Dilution series of standard peptides from.6 pm to 1 nm shows Median Fluorescence Intensity remains linear over 2-3 orders of magnitude, for quantitative power.

25 MILLIPLE MAP EpiQuant Products The MILLIPLE m a p EpiQuant platform unites two powerful technologies EpiQuant technology with the Luminex xmap platform to bring you the most advanced in quantitative cell signaling. Answering the challenges of multiplex cell signaling, MILLIPLE m a p EpiQuant technology enables you to: Design small- or large-plexed panels easily Customize your assay by selecting analytes you want to study Obtain quantitative results at picomolar sensitivities Measure phosphorylation concentrations of multiple sites on the same protein; all in one well Measure both total and phosphoprotein; all in the same well Utilize all the advantages and features associated with the MILLIPLE map assay format MILLIPLE m a p EpiQuant Sample Preparation Kit (Catalogue No. MPEQ-SP)* A sample prep kit containing all the reagents you need to prepare up to 5 ml of sample lysates, including: Cell lysis Protein denaturing Protein fragmentation *Kit required to run MILLIPLE map EpiQuant multiplex assays MILLIPLE m a p EpiQuant Phosphotyrosine Cell Signaling Panel 1 (Catalogue No. MPEQ-1K) All the components and reagents you need to simultaneously detect multiple phosphotyrosine sites in a small sample size The ability to choose analytes from different pathways to design a custom kit Standards included to obtain quantitative picomolar-range results Quality controls provided to qualify assay performance Analytically validated panels that yield consistent analyte profiles irrespective of plex size Standardized standard curve ranges across analytes and lots to ensure lot-to-lot consistency Panels that meet stringent manufacturing criteria to ensure batch-to-batch reproducibility Now Available! MILLIPLE map EpiQuant panel with 4 phosphotyrosine analytes Future panels will include other sets of total and phosphotyrosine-modified, phosphoserine-modified, and phosphothreonine-modified analytes. At the same time, we will be launching pathway-specific assays that enable you to explore at great depth such pathways as EGFR and p53.

26 Successful Multiplexing with MILLIPLE map EpiQuant Technology To overcome these multiplexing challenges, in addition to our MILLIPLE m a p cell signaling kits and MAPmate products, Millipore proudly introduces our new MILLIPLE m a p EpiQuant cell signaling portfolio major advance in the design and implementation of quantitative immunoassays. MILLIPLE m a p EpiQuant technology uses computational algorithms to identify unique, continuous linear sequences in proteins, generating antibodies against these specific sequences. Proteolytic digestion of your samples liberates these EpiQuant-bearing peptides, enabling you to simultaneously measure both total protein abundance and sitespecific phosphorylation at the peptide level, using EpiQuant antibodies. These peptides are then used to generate standard curves, which results in quantitative measurements with picomolar sensitivity. A unique feature of the EpiQuant sample processing protocol is fragmentation or digestion of the protein sample. This process has a number of benefits: Protein:protein interactions are disrupted A synthetic peptide can be used as a quantitative standard Specificity of the antibodies can be narrowed to a small linear peptide sequence Cross-reactivity with similar peptide sequences in other proteins can be predicted and avoided Residual enzymatic activity of the sample (e.g. phosphatases and proteases) is quenched

27 Multiplexing: Faster Answers, New Challenges Multiplexing, using the Luminex xmap platform, provides faster answers to cell signaling questions compared to traditional Western blots, mass spectrometry analyses and radioactive phosphorylation assays requiring large amounts of sample. Multiplexed immunoassays advance the understanding of intracellular communication by enabling the researcher to: Measure multiple total or phosphorylated sites along a signaling pathway, in a single sample Simultaneously measure sites in multiple pathways Conduct all necessary measurements using minimal sample volume However, multiplexing still presents some challenges there is a lack of phosphospecific antibodies that work well on the Luminex platform due to: Requirement for native protein conformation instead of recognition of denatured proteins on Western blots Complex buffer interactions with phosphoproteins Matrix and inter-antibody effects when multiplexed Resulting difficulties include: Lack of sufficient targets for pathway analysis Qualitative, not quantitative results Needing separate wells to analyze each phosphorylated site on a specific analyte, as well as total protein.

28 Data Sheet Product Selection Guide MILLIPLE MAP EpiQuant Technology and Panels Your Source for MORE: Flex-ability Quant-ability Plex-ability Site-ability Compat-ability