Multiplex Suspension Array Bio-Plex® Phosphoprotein Detection Explore the Depths Bio-Plex Phosphoprotein and Total Target Assays Available Assays Assays Phosphoprotein Total Akt Signaling Akt (Ser473) l l GSK-3a/b (Ser21/Ser9) l Cell Cycle/Checkpoint Control p53 (Ser15) l l new p53 (Ser37) l new l new p53 (Ser46) l new l new Chromatin Regulation/Acetylation Histone H3 (Ser10) l Immunology/Inflammation IkB-a (Ser32/Ser36) l l NF-kB p65 (Ser536) l STAT2 (Tyr689) l STAT3 (Ser727) l new STAT3 (Tyr705) l STAT6 (Tyr641) l Tyk2 (Tyr1054/Tyr1055) l new Glucose/Energy Metabolism IGF-IR (Tyr1131) l new IR-b (Tyr1146) l new IRS-1 (Ser636/Ser639) l MAP Kinase Signaling ATF-2 (Thr71) l l c-Jun (Ser63) l l ERK1 (Thr202/Tyr204) l ERK2 (Thr185/Tyr187) l l ERK1/2 (Thr202/Tyr204, Thr185/Tyr187) l l HSP27 (Ser78) l l JNK (Thr183/Tyr185) l l MEK1 (Ser217/Ser221) l l p38 MAPK (Thr180/Tyr182) l l p90RSK (Thr359/Ser363) l l Neuroscience CREB (Ser133) l l TrkA (Tyr490) l Translational Control p70 S6 kinase (Thr421/Ser424) l S6 ribosomal protein (Ser235/Ser236) l Tyrosine Kinases Bcr-Abl (Tyr245) l new l new c-Abl (Tyr245) l new c-Abl (Tyr412) l new EGFR (Tyr) l PDGF receptor-b (Tyr751) l Src (Tyr416) l new * You can design an x-Plex assay for combinations of assays. For more information, go to www.bio-rad.com/bio-plex/x-plex/ or contact your local Bio-Rad sales representative. Multiplex Assays for Signal Transduction Research Navigate Signaling Pathways These assays make it possible to simultaneously examine multiple phosphorylation events involving upstream or downstream molecules in one or more cellular signaling pathways. Multiplexing thus enables you to maximize the information obtained from a single sample by permitting the analysis of more than one target protein at a time, with minimal sample volume requirements and minimal hands-on time. Whether you’re evaluating the phosphorylation states of specific phosphoproteins or the expression levels of total target proteins, it’s now quicker and easier to get the data you’re looking for. CST antibodies exclusively developed and validated for Bio-Plex phosphoprotein and total target assays. MAPK/ERK in growth and differentiation. Courtesy of CST. Chart Phosphorylation Events Bio-Plex phosphoprotein assays and Bio-Plex total target assays are bead-based multiplexable assays that detect the phosphorylation and expression of proteins in lysates derived from cell culture or tissue samples. These 96-well microplate-formatted assays allow profiling of the specific phosphorylation states and expression levels of multiple proteins using only two wells and as little as 25 µl of lysate per well. The Bio-Plex total target assay reports the abundance of the target protein in one well, while the Bio-Plex phosphoprotein assay reports the phosphorylation level of the same protein in a separate well. 15,000 35,000 30,000 12,000 25,000 25,000 20,000 9,000 20,000 15,000 15,000 10,000 MFI 6,000 10,000 5,000 3,000 0 p-c-Jun 5,000 p-ERK1/2 0 0 p-MEK1 p-GSK-3a/b HEK 293 NIH 3T3 p-p53 10 5 2.5 1.25 0.63 0.31 HeLa UV/HEK 293 /HeLa p-JNK HeLa UV/COS /HeLa /HeLa EGF/HEK 293 a /HeLa a a Total protein, µg a UV/COS -NGF/PC-12IL-6/U-937 NIH 3T3 PDGF/NIHb 3T3 IL-4/HeLa HEK 293 IL-4/HeLa IFN- IFN- IL-6/U-937 TNF- TNF- UV/HEK 293 -NGF/PC-12 EGF/HEK 293 b PDGF/NIH 3T3 Bio-Plex 6-plex phosphoprotein assay analysis. Comparison of Bio-Plex phosphoprotein assays with western blots. Left, detection Activation of phospho-JNK (n), p53 (n), GSK-3a/b (n), 15 of phosphorylated p53 (Ser ) in 12 samples from 6 cell lines with various treatments. MEK1 (n), ERK1/2 (n), and c-Jun (n) was attenuated in Right, detection of phosphorylated p53 in dose-response study of ultraviolet (UV)-treated HEK 293, NIH 3T3, PC-12, U-937, COS, and HeLa cells HT-29 cell lysates. Upper panels, Bio-Plex assays; lower panels, western blots. treated with different ligands or stress-related methods. Limitless Combinations Flexible and Convenient Formats We are committed to the continual introduction of Bio-Plex phosphoprotein and total target assays are new assays to increase your multiplexing options. available as singleplex or premixed multiplex assays. Cell Signaling Technology (CST) is now developing n Singleplex assays are designed to be flexible. You can highly specific antibodies exclusively for Bio-Rad’s use them individually to test for a single phosphorylation state-specific Bio-Plex assays. phosphoprotein at a time, or you can combine them This partnership combines Bio-Rad’s expertise in to create a multiplex assay to test for a specific set of bead-based assay development and CST’s expertise phosphoproteins in a single sample in antibody development. Check in with us often to stay up-to-date on our new releases! n Premixed multiplex assays are the more convenient format for repeat testing of a specific set of Leading to Results phosphoproteins: The coupled beads are premixed Bio-Rad’s phosphorylation state-specific Bio-Plex and quality-tested at Bio-Rad, as are the detection assays feature: antibodies. Custom-premixed assays are available n Highly specific antibodies exclusively developed and through our online x-Plex™ assay service at validated by CST www.bio-rad.com/bio-plex/x-plex/ n Higher-throughput 96-well microplate format Specifications* n Higher-specificity sandwich format that minimizes Reactive species Human, mouse, rat, monkey cross-reaction with other cellular proteins Matrices Cell culture lysate, tissue lysate 25 µl n Direct measurement of kinases activated within the cell Sample volume Limit of detection <10 µg/ml of total protein n Simplified sample preparation with the Bio-Plex Precision Intra-assay CV <15% cell lysis kit, a kit specifically developed to simplify Inter-assay CV <25% preparation of cell culture and tissue sample lysates. Multiplexing capability Yes Lysates prepared with this protocol can be analyzed Storage conditions 2–8°C for coupled beads and detection with any of the Bio-Plex assays or western blotting antibodies (request bulletin 3033) –20°C for lysates (shipped separately) Stability** 12 months * Values vary by assay. ** Check label for expiration date. What You Need for Phosphoprotein 1 2 3 Bio-Plex phosphoprotein assays and Bio-Plex cell lysis kit — includes cell Bio-Plex phosphoprotein detection Detection Bio-Plex total target assays — include lysis buffer, cell wash buffer, factor 1, reagent kit — includes assay buffer, coupled beads, detection antibodies, and and factor 2; essential for optimal lysis wash buffer, antibody diluent, resuspension lysates to test for specific proteins of of your cell culture or tissue samples. buffer, streptavidin-PE, 96-well filter plate, interest in lysate samples. sealing tape, and instructions required to prepare assays and acquire data. 12 28 57 99 12 12 28 57 99 28 57 Bio-Plex Immunoassay Workflow 99 12 28 57 99 For Phosphoprotein and Total Target Assays 12 28 57 99 0 Bio-Plex assays 10 min 1 Coupled Beads 3 1 A Bio-Plex assay uses a selection of beads with different spectral addresses, 2 each coupled to antibodies against a different target. are designed using 0 n 10 min Add coupled beads to wells of a 96-well plate 3 1 0 n Wash microscopic beads, each 10 min + 2 overnight 3 1 0 with a different color 2 10 min 0 3 101 min + 2 Samples overnight 3 1 20 Tissue or cell lysates (prepared with a Bio-Plex cell lysis kit) can code (spectral address) 40 min 2 be used with Bio-Plex assays. 3 0 1 10 min n Add samples to wells containing coupled beads 0 3 2 110 min + to permit discrimination n 0 overnight Incubate overnight (or 15–18 hr) 3 1 2 40 min n Wash 3 1 20 of individual assays. 0 20 min 10 min 2 3 0 1 3 101 min + Incubate 15–18 hr overnight 0 Beads are dyed with 3 2 140 min 2 3 Detection Antibodies 0 3 201 min 2 Biotin-labeled detection antibodies are specific for secondary epitopes on 3 1 different ratios of two 20 0 ~40 min each target. 10 min + 2 overnight n 3 0 1 Add detection antibodies to wells 3 401 min 0 n fluorophores and are 3 2 120 min Incubate 30 min 2 0 3 1 n 2 ~40 min Wash 3 1 thus classified into 100 2 0 40 min 2 0 3 201 min unique bead regions 0 Reporter 3 1~40 min 4 2 3 1 Fluorescently labeled streptavidin reporter binds to the biotin-labeled detection 2 (xMAP technology). antibodies. 2 0 n 20 min Add reporter to wells 0 3 ~401 min n Incubate 10 min 3 1 2 n Rinse 2 n Resuspend in assay buffer 0 ~40 min 3 1 12 28 57 99 2 5 Assay In the Bio-Plex array reader, a red classification laser and a green reporter laser illuminate individual beads to identify each bead’s spectral address and associated reporter signal. n Dyed beads are identified by their internal fluorescent signature (bead region) Bio-Plex phosphoprotein detection n Level of target bound to beads is indicated by intensity of reporter signal reagent kit — includes assay buffer, n wash buffer, antibody diluent, resuspension Multiplex data are reported simultaneously buffer, streptavidin-PE, 96-well filter plate, sealing tape, and instructions required to prepare assays and acquire data.