Phosphoprotein Detection
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- Jared Jordan
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1 Mutipex Suspesio Array Bio-Pex Phosphoprotei Detectio Expore the Depths
2 Bio-Pex Phosphoprotei ad Tota Target Assays Avaiabe Assays Assays Phosphoprotei Tota Akt Sigaig Akt (Ser 47 ) GSK-a/b (Ser /Ser 9 ) Ce Cyce/Checkpoit Cotro p5 (Ser 5 ) ew p5 (Ser 7 ) ew ew p5 (Ser 46 ) ew ew Chromati Reguatio/Acetyatio Histoe H (Ser ) Immuoogy/Ifammatio IkB-a (Ser /Ser 6 ) NF-kB p65 (Ser 56 ) STAT (Tyr 689 ) STAT (Ser 77 ) STAT (Tyr 75 ) STAT6 (Tyr 64 ) Tyk (Tyr 54 /Tyr 55 ) Gucose/Eergy Metaboism IGF-IR (Tyr ) IR-b (Tyr 46 ) IRS- (Ser 66 /Ser 69 ) MAP Kiase Sigaig ATF- (Thr 7 ) c-ju (Ser 6 ) ERK (Thr /Tyr 4 ) ERK (Thr 85 /Tyr 87 ) ERK/ (Thr /Tyr 4, Thr 85 /Tyr 87 ) HSP7 (Ser 78 ) JNK (Thr 8 /Tyr 85 ) MEK (Ser 7 /Ser ) p8 MAPK (Thr 8 /Tyr 8 ) p9rsk (Thr 59 /Ser 6 ) Neurosciece CREB (Ser ) TrkA (Tyr 49 ) Trasatioa Cotro p7 S6 kiase (Thr 4 /Ser 44 ) S6 ribosoma protei (Ser 5 /Ser 6 ) Tyrosie Kiases Bcr-Ab (Tyr 45 ) ew ew c-ab (Tyr 45 ) ew c-ab (Tyr 4 ) ew EGFR (Tyr) PDGF receptor-b (Tyr 75 ) Src (Tyr 46 ) ew * You ca desig a x-pex assay for combiatios of assays. For more iformatio, go to or cotact your oca Bio-Rad saes represetative. ew ew ew ew
3 Mutipex Assays for Siga Trasductio Research Navigate Sigaig Pathways These assays make it possibe to simutaeousy examie mutipe phosphoryatio evets ivovig upstream or dowstream moecues i oe or more ceuar sigaig pathways. Mutipexig thus eabes you to maximize the iformatio obtaied from a sige sampe by permittig the aaysis of more tha oe target protei at a time, with miima sampe voume requiremets ad miima hads-o time. Whether you re evauatig the phosphoryatio states of specific phosphoproteis or the expressio eves of tota target proteis, it s ow quicker ad easier to get the data you re ookig for. CST atibodies excusivey deveoped ad vaidated for Bio-Pex phosphoprotei ad tota target assays. MAPK/ERK i growth ad differetiatio. Courtesy of CST. Chart Phosphoryatio Evets Bio-Pex phosphoprotei assays ad Bio-Pex tota target assays are bead-based mutipexabe assays that detect the phosphoryatio ad expressio of proteis i ysates derived from ce cuture or tissue sampes. These 96-we micropate-formatted assays aow profiig of the specific phosphoryatio states ad expressio eves of mutipe proteis usig oy two wes ad as itte as 5 µ of ysate per we. The Bio-Pex tota target assay reports the abudace of the target protei i oe we, whie the Bio-Pex phosphoprotei assay reports the phosphoryatio eve of the same protei i a separate we.
4 MFI 5,, 9, 6,, HEK 9 UV/HEK 9 EGF/HEK 9 NIH T PDGF/NIH T b-ngf/pc- IL-6/U-97 HeLa IFN-a/HeLa UV/COS IL-4/HeLa TNF-a/HeLa 5,, 5,, 5,, 5, Tota protei, µg Compariso of Bio-Pex phosphoprotei assays with wester bots. Left, detectio of phosphoryated p5 (Ser 5 ) i sampes from 6 ce ies with various treatmets. Right, detectio of phosphoryated p5 i dose-respose study of utravioet (UV)-treated HT-9 ce ysates. Upper paes, Bio-Pex assays; ower paes, wester bots. 5,, 5,, 5, HEK 9 UV/HEK 9 EGF/HEK 9 NIH T PDGF/NIH T b-ngf/pc- UV/COS HeLa IFN-a/HeLa IL-4/HeLa TNF-a/HeLa IL-6/U-97 p-c-ju p-erk/ p-mek p-gsk-a/b p-p5 p-jnk Bio-Pex 6-pex phosphoprotei assay aaysis. Activatio of phospho-jnk (), p5 (), GSK-a/b (), MEK (), ERK/ (), ad c-ju () was atteuated i HEK 9, NIH T, PC-, U-97, COS, ad HeLa ces treated with differet igads or stress-reated methods. Limitess Combiatios We are committed to the cotiua itroductio of ew assays to icrease your mutipexig optios. Ce Sigaig Techoogy (CST) is ow deveopig highy specific atibodies excusivey for Bio-Rad s phosphoryatio state-specific Bio-Pex assays. This partership combies Bio-Rad s expertise i bead-based assay deveopmet ad CST s expertise i atibody deveopmet. Check i with us ofte to stay up-to-date o our ew reeases! Leadig to Resuts Bio-Rad s phosphoryatio state-specific Bio-Pex assays feature: Highy specific atibodies excusivey deveoped ad vaidated by CST Higher-throughput 96-we micropate format Higher-specificity sadwich format that miimizes cross-reactio with other ceuar proteis Direct measuremet of kiases activated withi the ce Simpified sampe preparatio with the Bio-Pex ce ysis kit, a kit specificay deveoped to simpify preparatio of ce cuture ad tissue sampe ysates. Lysates prepared with this protoco ca be aayzed with ay of the Bio-Pex assays or wester bottig (request bueti ) Fexibe ad Coveiet Formats Bio-Pex phosphoprotei ad tota target assays are avaiabe as sigepex or premixed mutipex assays. Sigepex assays are desiged to be fexibe. You ca use them idividuay to test for a sige phosphoprotei at a time, or you ca combie them to create a mutipex assay to test for a specific set of phosphoproteis i a sige sampe Premixed mutipex assays are the more coveiet format for repeat testig of a specific set of phosphoproteis: The couped beads are premixed ad quaity-tested at Bio-Rad, as are the detectio atibodies. Custom-premixed assays are avaiabe through our oie x-pex assay service at Specificatios* Reactive species Huma, mouse, rat, mokey Matrices Ce cuture ysate, tissue ysate Sampe voume 5 µ Limit of detectio < µg/m of tota protei Precisio Itra-assay CV <5% Iter-assay CV <5% Mutipexig capabiity Yes Storage coditios 8 C for couped beads ad detectio atibodies C for ysates (shipped separatey) Stabiity** moths * Vaues vary by assay. ** Check abe for expiratio date. What You Need for Phosphoprotei Detectio Bio-Pex phosphoprotei assays ad Bio-Pex tota target assays icude couped beads, detectio atibodies, ad ysates to test for specific proteis of iterest i ysate sampes. Bio-Pex ce ysis kit icudes ce ysis buffer, ce wash buffer, factor, ad factor ; essetia for optima ysis of your ce cuture or tissue sampes. Bio-Pex phosphoprotei detectio reaget kit icudes assay buffer, wash buffer, atibody diuet, resuspesio buffer, streptavidi-pe, 96-we fiter pate, seaig tape, ad istructios required to prepare assays ad acquire data.
5 Bio-Pex Immuoassay Workfow For Phosphoprotei ad Tota Target Assays Bio-Pex assays are desiged usig microscopic beads, each with a differet coor of idividua assays. Beads are dyed with differet ratios of two fuorophores ad are thus cassified ito uique bead regios to permit discrimiatio (xmap techoogy). mi Add couped beads to wes of a 96-we pate Wash mi + overight mi mi + overight 4 mi mi mi + overight mi 4 Add sampes to wes cotaiig couped beads Icubate overight (or 5 8 hr) Wash mi mi Icubate 5 8 hr mi + overight 4 mi mi ~4 mi mi + overight 4 mi Detectio Atibodies Bioti-abeed detectio atibodies are specific for secodary epitopes o each target. mi ~4 mi Add detectio atibodies to wes Icubate mi Wash 4 mi mi ~4 4 mi Reporter Fuorescety abeed streptavidi reporter bids to the bioti-abeed detectio atibodies. mi Add reporter to wes ~4 mi Icubate mi Rise Resusped i assay buffer ~4 mi Sampes Tissue or ce ysates (prepared with a Bio-Pex ce ysis kit) ca be used with Bio-Pex assays. Couped Beads A Bio-Pex assay uses a seectio of beads with differet spectra addresses, each couped to atibodies agaist a differet target. code (spectra address) mi 8 5 Assay I the Bio-Pex array reader, a red cassificatio aser ad a gree reporter aser iumiate idividua beads to idetify each bead s spectra address ad associated reporter siga. Dyed beads are idetified by their itera fuorescet sigature (bead regio) Leve of target boud to beads is idicated by itesity of reporter siga Mutipex data are reported simutaeousy
6 Orderig Iformatio Cataog # Descriptio Bio-Pex Phosphoprotei Sigepex Assays,* x 96-we 7-V75 Bio-Pex Phospho-Akt (Ser 47 ) Assay 7-V6 Bio-Pex Phospho-ATF- (Thr 7 ) Assay 7-V745 Bio-Pex Phospho-Bcr-Ab (Tyr 45 ) Assay 7-V7545 Bio-Pex Phospho-c-Ab (Tyr 45 ) Assay 7-V7745 Bio-Pex Phospho-c-Ab (Tyr 4 ) Assay 7-V456 Bio-Pex Phospho-c-Ju (Ser 6 ) Assay 7-V69 Bio-Pex Phospho-CREB (Ser ) Assay 7-V** Bio-Pex Phospho-EGFR (Tyr) Assay 7-V98 Bio-Pex Phospho-ERK (Thr /Tyr 4 ) Assay 7-V48 Bio-Pex Phospho-ERK (Thr 85 /Tyr 87 ) Assay 7-V8 Bio-Pex Phospho-ERK/ (Thr /Tyr 4, Thr 85 /Tyr 87 ) Assay 7-V8 Bio-Pex Phospho-GSK-a/b (Ser /Ser 9 ) Assay 7-V4777 Bio-Pex Phospho-Histoe H (Ser ) Assay 7-V455 Bio-Pex Phospho-HSP7 (Ser 78 ) Assay 7-V74 Bio-Pex Phospho-IGF-IR (Tyr ) Assay 7-V758 Bio-Pex Phospho-IkB-a (Ser /Ser 6 ) Assay 7-V794 Bio-Pex Phospho-IR-b (Tyr 46 ) Assay 7-V56 Bio-Pex Phospho-IRS- (Ser 66 /Ser 69 ) Assay 7-V4 Bio-Pex Phospho-JNK (Thr 8 /Tyr 85 ) Assay 7-V54 Bio-Pex Phospho-MEK (Ser 7 /Ser ) Assay 7-V497 Bio-Pex Phospho-NF-kB p65 (Ser 56 ) Assay 7-V6 Bio-Pex Phospho-p8 MAPK (Thr 8 /Tyr 8 ) Assay 7-V55 Bio-Pex Phospho-p5 (Ser 5 ) Assay 7-V85 Bio-Pex Phospho-p5 (Ser 7 ) Assay 7-V85 Bio-Pex Phospho-p5 (Ser 46 ) Assay 7-V455 Bio-Pex Phospho-p7 S6 Kiase (Thr 4 /Ser 44 ) Assay 7-V55 Bio-Pex Phospho-p9RSK (Thr 59 /Ser 6 ) Assay 7-V59 Bio-Pex Phospho-PDGF Receptor-b (Tyr 75 ) Assay 7-V674 Bio-Pex Phospho-Src (Tyr 46 ) Assay 7-V4** Bio-Pex Phospho-S6 Ribosoma Protei (Ser 5 /Ser 6 ) Assay 7-V7 Bio-Pex Phospho-STAT (Tyr 689 ) Assay 7-V695 Bio-Pex Phospho-STAT (Ser 77 ) Assay 7-V55 Bio-Pex Phospho-STAT (Tyr 75 ) Assay 7-V7 Bio-Pex Phospho-STAT6 (Tyr 64 ) Assay 7-V97 Bio-Pex Phospho-TrkA (Tyr 49 ) Assay 7-V65 Bio-Pex Phospho-Tyk (Tyr 54 /Tyr 55 ) Assay Bio-Pex Premixed Phosphoprotei Assay Paes X7AB Bio-Pex Phospho -Pex Pae, x 96-we, icudes couped beads, detectio atibodies, ad ysates for the detectio of phosphoryated GSK-a/b, p7 S6 kiase, ad STAT X7CLDZ Bio-Pex Phospho -Pex Pae, x 96-we, icudes couped beads, detectio atibodies, ad ysates for the detectio of phosphoryated ATF-, NF-kB p65, ad p9rsk X7RD Bio-Pex Phospho 5-Pex Pae, x 96-we, icudes couped beads, detectio atibodies, ad ysates for the detectio of phosphoryated Akt, ERK/, IkB-a, JNK, ad p8 MAPK * For mutipexig capabiities of specific assay combiatios, request bueti 548, or visit us o the Web at ** This assay caot be mutipexed. Cataog # Descriptio Bio-Pex Tota Target Sigepex Assays* 7-V75 Bio-Pex Tota Akt Assay, x 96-we 7-V6 Bio-Pex Tota ATF- Assay, x 96-we 7-V745 Bio-Pex Tota Bcr-Ab Assay, x 96-we 7-V456 Bio-Pex Tota c-ju Assay, x 96-we 7-V69 Bio-Pex Tota CREB Assay, x 96-we 7-V48 Bio-Pex Tota ERK Assay, x 96-we 7-V8 Bio-Pex Tota ERK/ Assay, x 96-we 7-V455 Bio-Pex Tota HSP7 Assay, x 96-we 7-V758 Bio-Pex Tota IkB-a Assay, x 96-we 7-V4 Bio-Pex Tota JNK Assay, x 96-we 7-V54 Bio-Pex Tota MEK Assay, x 96-we 7-V6 Bio-Pex Tota p8 MAPK Assay, x 96-we 7-V55 Bio-Pex Tota p5 Assay, x 96-we 7-V55 Bio-Pex Tota p9rsk Assay, x 96-we Bio-Pex Premixed Tota Target Assay Paes X9TK Bio-Pex Tota 5-Pex Pae, x 96-we, icudes couped beads, detectio atibodies, ad ysates for the detectio of Akt, ERK/, IkB-a, JNK, ad p8 MAPK Bio-Pex Reaget Kits 7-44 Bio-Pex Phosphoprotei Detectio Reaget Kit, x 96-we 7-45 Bio-Pex Phosphoprotei Detectio Reaget Kit, x 96-we Bio-Pex Sampe Preparatio Kits 7-4 Bio-Pex Ce Lysis Kit, x 96-we 7-4 Bio-Pex Ce Lysis Kit, x 96-we x-pex Assay Service These assays are custom-premixed ad quaity-tested at Bio-Rad. Seect your desired combiatio of Bio-Pex phosphoprotei or tota target assays oie at For more iformatio, request bueti 95. Ce Sigaig Techoogy ad the Ce Sigaig Techoogy ogo are trademarks of Ce Sigaig Techoogy. xmap is a trademark of Lumiex Corporatio. The Bio-Pex suspesio array system icudes fuorescety abeed microspheres ad istrumetatio icesed to Bio-Rad Laboratories, Ic. by the Lumiex Corporatio. Bio-Rad Laboratories, Ic. Life Sciece Group Web site USA 8 4BIORAD Austraia Austria Begium Brazi Caada Chia Czech Repubic Demark 44 5 Fiad 9 84 Frace Germay Greece Hog Kog Hugary Idia Israe Itay 9 69 Japa Korea Mexico The Netherads New Zeaad Norway 8 4 Poad 48 Portuga Russia Sigapore South Africa Spai Swede Switzerad Taiwa Uited Kigdom 88 Bueti 9 US/EG Rev F Sig 6
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