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    Pacific Blue™ Mouse anti-ERK1/2 (pT202/pY204)
    Pacific Blue™ Mouse anti-ERK1/2 (pT202/pY204)
    Analysis of Erk1/2 (pT202/pY204) in human peripheral blood lymphocytes. Human peripheral blood mononuclear cells (PBMC) were either left untreated (unshaded) or treated (shaded) with 400 nM phorbol 12-myristate 13-acetate (PMA) (Sigma, Cat. No. P8139) for 15 minutes at 37°C. The PBMC were fixed with BD Cytofix™ buffer (Cat. No. 554655) for 10 minutes at 37°C, permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for 30 minutes and were then stained with Pacific Blue™ Mouse anti-ERK1/2 (pT202/pY204). For data analysis, lymphocytes were selected by scatter profile. Flow cytometry was performed on a BD FACSCanto™ II flow cytometry system.
    Pacific Blue™ Mouse anti-ERK1/2 (pT202/pY204) Pacific Blue™ Mouse anti-ERK1/2 (pT202/pY204)
    Analysis of Erk1/2 (pT202/pY204) in human peripheral blood lymphocytes. Human peripheral blood mononuclear cells (PBMC) were either left untreated (unshaded) or treated (shaded) with 400 nM phorbol 12-myristate 13-acetate (PMA) (Sigma, Cat. No. P8139) for 15 minutes at 37°C. The PBMC were fixed with BD Cytofix™ buffer (Cat. No. 554655) for 10 minutes at 37°C, permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for 30 minutes and were then stained with Pacific Blue™ Mouse anti-ERK1/2 (pT202/pY204). For data analysis, lymphocytes were selected by scatter profile. Flow cytometry was performed on a BD FACSCanto™ II flow cytometry system.
    Product Details
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    BD Phosflow™
    p44/42 MAPK; Extracellular signal-Regulated Kinase 1/2 (pT202/Y204)
    Human (QC Testing)
    Mouse IgG1
    Phosphorylated Rat ERK1 (T202/Y204) Peptide
    Intracellular staining (flow cytometry) (Routinely Tested)
    20 µl
    AB_1645294
    Aqueous buffered solution containing BSA, protein stabilizer, and ≤0.09% sodium azide.
    RUO


    Preparation And Storage

    Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody is conjugated to Pacific Blue™ under optimum conditions, and unreacted Pacific Blue™ was removed.
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    Recommended Assay Procedures

    This antibody conjugate is suitable for intracellular staining of human whole blood (using BD Phosflow™ Lyse/Fix Buffer) and peripheral blood mononuclear cells (using BD Cytofix™ Fixation Buffer).  Any of the three BD Phosflow™ permeabilization buffers may be used.

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    Product Notices

    1. This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
    2. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
    3. Pacific Blue™ has a maximum absorption of 416 nm and maximum emission of 451 nm. Before staining with this reagent, please confirm that your flow cytometer is capable of exciting the fluorochrome and discriminating the resulting fluorescence.
    4. Pacific Blue™ is a trademark of Molecular Probes, Inc., Eugene, OR.
    5. The Alexa Fluor®, Pacific Blue™, and Cascade Blue® dye antibody conjugates in this product are sold under license from Molecular Probes, Inc. for research use only, excluding use in combination with microarrays, or as analyte specific reagents. The Alexa Fluor® dyes (except for Alexa Fluor® 430), Pacific Blue™ dye, and Cascade Blue® dye are covered by pending and issued patents.
    6. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
    7. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
    8. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
    9. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
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    560314 Rev. 2
    Antibody Details
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    20A

    The members of the Mitogen-Activated Protein Kinase (MAPK) family are components of a key signal transduction cascade that links events at the cell surface to responses in the nucleus. The signaling cascade is found in species as varied as yeast and humans, with many of the proteins being well conserved. In mammals the most widely studied members of the cascade are the Extracellular signal-Regulated Kinases, ERK1 (p44 MAPK) and ERK2 (p42 MAPK).  ERK1 and ERK2 share 85% homology and are activated by extracellular signals such as growth factors, hormones, and phorbol esters. Activation occurs through a series of phosphorylations by kinases activating other kinases and eventually leading to phosphorylation of the ERKs. Growth factor stimulation leads to activation of Ras and Raf, leading to phosphorylation of MEK1 (MAPK/ERK kinase) which, in turn, activates the ERKs via dual phosphorylation. Once activated, the ERKs phosphorylate other cytoplasmic signalling molecules, cell-surface receptors, microtubule-associated proteins, and transcription factors in the nucleus. Thus, the active ERK has myriad downstream effectors that implicate it in the control of cell proliferation and differentiation, as well as regulation of the cytoskeleton. Furthermore, studies have shown that elevated ERK activity is associated with some cancers.

    The 20A monoclonal antibody recognizes the phosphorylated threonine 202 and tyrosine 204 (pT202/pY204) of human ERK1 and pT184/pY186 of human ERK2. The orthologous phosphorylation sites in murine ERK1 and ERK2 are T203/Y205 and T183/Y185.

    560314 Rev. 2
    Format Details
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    Pacific Blue
    Pacific Blue™ is part of the BD violet family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 404-nm and an emission maximum (Em Max) at 455-nm. Pacific Blue is designed to be excited by the violet laser (405-nm) and detected using an optical filter centered near 450-nm (e.g., a 450/50-nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
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    Pacific Blue
    Violet 405 nm
    404 nm
    455 nm
    560314 Rev.2
    Citations & References
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    Development References (10)

    1. Boulton TG, Cobb MH. Identification of multiple extracellular signal-regulated kinases (ERKs) with antipeptide antibodies. Cell Regul. 1991; 2(5):357-371. (Biology). View Reference
    2. Clark EA, Hynes RO. Ras activation is necessary for integrin-mediated activation of extracellular signal-regulated kinase 2 and cytosolic phospholipase A2 but not for cytoskeletal organization. J Biol Chem. 1996; 271(25):14814-14818. (Biology). View Reference
    3. Cobb MH, Boulton TG, Robbins DJ. Extracellular signal-regulated kinases: ERKs in progress. Cell Regul. 1991; 2:965-978. (Biology). View Reference
    4. Irish JM, Czerwinski DK, Nolan GP, Levy R. Altered B-cell receptor signaling kinetics distinguish human follicular lymphoma B cells from tumor-infiltrating nonmalignant B cells. Blood. 2006; 108(9):3135-3142. (Clone-specific: Flow cytometry). View Reference
    5. Irish JM, Czerwinski DK, Nolan GP, Levy R. Kinetics of B cell receptor signaling in human B cell subsets mapped by phosphospecific flow cytometry. J Immunol. 2006; 177(3):1581-1589. (Clone-specific: Flow cytometry). View Reference
    6. Kim SC, Hahn JS, Min YH, Yoo NC, Ko YW, Lee WJ. Constitutive activation of extracellular signal-regulated kinase in human acute leukemias: combined role of activation of MEK, hyperexpression of extracellular signal-regulated kinase, and downregulation of a phosphatase, PAC1. Blood. 1999; 93(11):3893-3899. (Clone-specific: In vitro kinase assay, Western blot). View Reference
    7. Krutzik PO, Nolan GP. Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling. Nat Methods. 2006; 3(5):361-368. (Clone-specific: Flow cytometry). View Reference
    8. Krutzik PO, Nolan GP. Intracellular phospho-protein staining techniques for flow cytometry: monitoring single cell signaling events. Cytometry A. 2003; 55(2):61-70. (Clone-specific: Flow cytometry). View Reference
    9. Sivaraman VS, Wang H, Nuovo GJ, Malbon CC. Hyperexpression of mitogen-activated protein kinase in human breast cancer. J Clin Invest. 1997; 99(7):1478-1483. (Biology). View Reference
    10. Treisman R. Regulation of transcription by MAP kinase cascades. Curr Opin Cell Biol. 1996; 8:205-215. (Biology). View Reference
    View All (10) View Less
    560314 Rev. 2

    Please refer to Support Documents for Quality Certificates


    Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described


    Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims.  Comparisons are not made against non-BD technologies, unless otherwise noted.

    For Research Use Only. Not for use in diagnostic or therapeutic procedures.

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