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PE Mouse Anti-Stat5 (pY694)
PE Mouse Anti-Stat5 (pY694)

Flow cytometric analysis of Stat5 (pY694) in human lysed whole blood.  Whole blood was either left untreated (dashed line histogram) or treated with 100 ng/mL Recombinant Human IL-2 for 15 minutes at 37°C (shaded histogram; Cat. No. 554603). The samples were lysed and fixed with 1X Lyse/Fix Buffer 5X (Cat. No. 558049) for 10 minutes at 37°C, permeabilized (Perm Buffer III; Cat. No. 558050) on ice for 30 minutes and were then stained with PE Mouse Anti-Stat5 (pY694), (Cat. No. 612567). The fluorescence histograms showing STAT5 (PY694) or Ig isotype control staining were derived from gated events with the forward and side light-scattering characteristics of viable lymphocytes. Flow cytometry was performed on a BD FACSCalibur™.

Flow cytometric analysis of Stat5 (pY694) in human lysed whole blood.  Whole blood was either left untreated (dashed line histogram) or treated with 100 ng/mL Recombinant Human IL-2 for 15 minutes at 37°C (shaded histogram; Cat. No. 554603). The samples were lysed and fixed with 1X Lyse/Fix Buffer 5X (Cat. No. 558049) for 10 minutes at 37°C, permeabilized (Perm Buffer III; Cat. No. 558050) on ice for 30 minutes and were then stained with PE Mouse Anti-Stat5 (pY694), (Cat. No. 612567). The fluorescence histograms showing STAT5 (PY694) or Ig isotype control staining were derived from gated events with the forward and side light-scattering characteristics of viable lymphocytes. Flow cytometry was performed on a BD FACSCalibur™.

Product Details
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BD Phosflow™
Signal transducer and activator of transcription 5; MGF; MPF
Human (QC Testing), Mouse, Rat, Sheep (Predicted)
Mouse IgG1, κ
Phosphorylated Human Phosphorylated Stat5 Peptide
Intracellular staining (flow cytometry) (Routinely Tested)
20 µl
AB_399858
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 was conjugated with R-PE under optimum conditions, and unconjugated antibody and free PE were removed.

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 or BD Phosflow Fix Buffer I).

This mAb was characterized by flow cytometry (Flow) and western blot analysis (WB) using these model systems:

Method                        Species                Cells                        Treatment        Fixation                                Perm buffer                Result

Flow                                Human                PBMC                        IL-2                        Fixation Buffer                III                                Positive Staining

Flow                                Human                PBMC                        IL-2                        Fixation Buffer                I or II                        Unsatisfactory

Flow                                Human                Whole Blood                IL-2                        Lyse/Fix                                III                                Positive Staining

Flow                                Human                Whole Blood                IL-2                        Lyse/Fix                                 I or II                        Unsatisfactory

Flow                Human                TF-1 cells                GM-CSF                Fixation Buffer                III                                Positive Staining

Flow                Human                TF-1 cells                GM-CSF                Fixation Buffer                I or II                        Unsatisfactory

WB                        Human                A431 Cell Lysate        EGF                        Not Applicable                Not Applicable        92 kDa

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. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  3. 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.
  4. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  5. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
612567 Rev. 9
Antibody Details
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47/Stat5(pY694)

Stat (Signal transducer and activators of transcription) proteins mediate the biological activity of cytokines, including interleukins, interferons, erythropoietin, and growth factors. Ligand-receptor interaction activates constitutively-associated JAK family kinases and subsequent recruitment/activation of Stat proteins by tyrosine phosphorylation. Active Stat proteins then move to the nucleus to promote transcription of cytokine-inducible genes. Seven Stat proteins have been cloned, each of which is differentially expressed and/or activated in a cytokine-specific and cell type-specific manner. Stat5 has been characterized and shown to be encoded by two separate genes, Stat5a and Stat5b, which share over 90% identity at the amino acid level. Stat5a has been shown to be involved in lactogenesis and mammary development, while Stat5b has been shown to be involved in growth hormone signaling and liver gene expression. Both Stat5a and Stat5b are involved in IL-2 induced peripheral T cell proliferation. The peptide hormone, prolactin, binds to the prolactin receptor (PRLR) to initiate the lactogenic response. There are at least three forms of PRLR; however, only the long form activates the 92-kDa Stat5 protein by inducing phosphorylation at Y694. Once phosphorylated, Stat5 becomes an essential transcription factor which binds to the β-casein gene promoter. The presence of an SH2 domain within Stat5 suggests that it may directly interact with protein tyrosine kinases (PTKs) such as JAK2.

The 47 monoclonal antibody recognizes the phosphorylated Y694 of Stat5a. The homologous phosphorylation site in Stat5b is Y699.

612567 Rev. 9
Format Details
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PE
R-Phycoerythrin (PE), is part of the BD family of Phycobiliprotein dyes. This fluorochrome is a multimeric fluorescent phycobiliprotein with excitation maximum (Ex Max) of 496 nm and 566 nm and an emission maximum (Em Max) at 576 nm. PE is designed to be excited by the Blue (488 nm), Green (532 nm) and Yellow-Green (561 nm) lasers and detected using an optical filter centered near 575 nm (e.g., a 575/26-nm bandpass filter). As PE is excited by multiple lasers, this can result in cross-laser excitation and fluorescence spillover on instruments with various combinations of Blue, Green, and Yellow-Green lasers. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
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PE
Yellow-Green 488 nm, 532 nm, 561 nm
496 nm, 566 nm
576 nm
612567 Rev.9
Citations & References
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Development References (5)

  1. Bromberg J, Darnell JE. The role of STATs in transcriptional control and their impact on cellular function. Oncogene. 2000; 19(21):2468-2473. (Biology). View Reference
  2. Gouilleux F, Wakao H, Mundt M, Groner B. Prolactin induces phosphorylation of Tyr694 of Stat5 (MGF), a prerequisite for DNA binding and induction of transcription. EMBO J. 1994; 13(18):4361-4369. (Biology). View Reference
  3. Liu KD, Gaffen SL, Goldsmith MA. JAK/STAT signaling by cytokine receptors. Curr Opin Immunol. 1998; 10(3):271-278. (Biology). View Reference
  4. Van De Wiele CJ, Marino JH, Murray BW, Vo SS, Whetsell ME, Teague TK. Thymocytes between the -Selection and Positive Selection Checkpoints Are Nonresponsive to IL-7 as Assessed by STAT-5 Phosphorylation. J Immunol. 2004; 172(7):4235-4244. (Biology). View Reference
  5. Wakao H, Gouilleux F, Groner B. Mammary gland factor (MGF) is a novel member of the cytokine regulated transcription factor gene family and confers the prolactin response. EMBO J. 1994; 13(9):2182-2191. (Biology). View Reference
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612567 Rev. 9

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