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RY586 Mouse Anti-Stat5 (pY694)
RY586 Mouse Anti-Stat5 (pY694)
Flow cytometric analysis of Stat5 (PY694) expression in human CD4+ T lymphocytes. Untreated (dashed line histogram) or treated (solid line histogram) peripheral blood mononuclear cells (PBMC) from the BD Phosflow™ T Cell Kit Lyophilized Cells (Catalog No. 560760) were reconstituted in BD Pharmingen™ Stain Buffer (Cat. No. 554656) for 15 minutes at room temperature. These cells were fixed, permeabilized, and lyophilized PBMC that were either untreated or treated with stimulators [including phorbol 12-myristate 13-acetate (PMA), interferon-alpha (IFN-α), interleukin-2 (IL-2), IL-4, and IL-6]. The reconstituted cells were then washed and stained with BD Pharmingen™ APC Mouse Anti-Human CD4 antibody (Cat. No. 566915) and BD Phosflow™ RY586 Mouse Anti-Human Stat5 (pY694) antibody (Cat. No. 568145/568146). Histograms showing Stat5 (pY694) expression were derived from CD4-positive gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ Flow Cytometer System and FlowJo™ software.​
Flow cytometric analysis of Stat5 (PY694) expression in human CD4+ T lymphocytes. Untreated (dashed line histogram) or treated (solid line histogram) peripheral blood mononuclear cells (PBMC) from the BD Phosflow™ T Cell Kit Lyophilized Cells (Catalog No. 560760) were reconstituted in BD Pharmingen™ Stain Buffer (Cat. No. 554656) for 15 minutes at room temperature. These cells were fixed, permeabilized, and lyophilized PBMC that were either untreated or treated with stimulators [including phorbol 12-myristate 13-acetate (PMA), interferon-alpha (IFN-α), interleukin-2 (IL-2), IL-4, and IL-6]. The reconstituted cells were then washed and stained with BD Pharmingen™ APC Mouse Anti-Human CD4 antibody (Cat. No. 566915) and BD Phosflow™ RY586 Mouse Anti-Human Stat5 (pY694) antibody (Cat. No. 568145/568146). Histograms showing Stat5 (pY694) expression were derived from CD4-positive gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ Flow Cytometer System and FlowJo™ software.​
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)
5 µl
Aqueous buffered solution containing ≤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 to the dye under optimum conditions and unreacted dye was removed.

Recommended Assay Procedures

BD® CompBeads can be used as surrogates to assess fluorescence spillover (compensation). When fluorochrome conjugated antibodies are bound to BD® CompBeads, they have spectral properties very similar to cells. However, for some fluorochromes there can be small differences in spectral emissions compared to cells, resulting in spillover values that differ when compared to biological controls. It is strongly recommended that when using a reagent for the first time, users compare the spillover on cells and BD® CompBeads to ensure that BD® CompBeads are appropriate for your specific cellular application.

Product Notices

  1. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  2. 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.
  3. 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).
  4. An isotype control should be used at the same concentration as the antibody of interest.
  5. CF™ is a trademark of Biotium, Inc.
  6. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
  7. Human donor specific background has been observed in relation to the presence of anti-polyethylene glycol (PEG) antibodies, developed as a result of certain vaccines containing PEG, including some COVID-19 vaccines. We recommend use of BD Horizon Brilliant™ Stain Buffer in your experiments to help mitigate potential background. For more information visit https://www.bdbiosciences.com/en-us/support/product-notices.
  8. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
568146 Rev. 2
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.

568146 Rev. 2
Format Details
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RY586
The BD Horizon RealYellow™ 586 (RY586) Dye is part of the BD family of yellow-green dyes. It is a small organic fluorochrome with an excitation maximum (Ex Max) at 565-nm and an emission maximum (Em Max) at 586-nm. Driven by BD innovation, RY586 can be used on both spectral and conventional cytometers and is designed to be excited by the Yellow-Green laser (561-nm) with minimal excitation by the 488-nm Blue laser. For conventional instruments equipped with a Yellow-Green laser (561-nm), RY586 can be used as an alternative to PE and we recommend using an optical filter centered near 586-nm (eg, a 586/15-nm bandpass filter). For spectral instruments equipped with a Yellow-Green laser (561-nm), it can be used in conjunction with PE. Compared to PE, RY586 is similar in brightness, minimal spillover into Blue detectors, and increased spillover into the 610/20-nm (PE-CF594) detector. Please ensure that your instrument configuration (lasers and optical filters) is appropriate for this dye.
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RY586
Yellow-Green 561 nm
564 nm
586 nm
568146 Rev.2
Citations & References
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View product citations for antibody "568146" on CiteAb

Development References (8)

  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. Johnston RJ, Choi YS, Diamond JA, Yang JA, Crotty S. STAT5 is a potent negative regulator of TFH cell differentiation. J Exp Med. 2012; 209(2):243-250. (Clone-specific: Flow cytometry). View Reference
  4. Liu KD, Gaffen SL, Goldsmith MA. JAK/STAT signaling by cytokine receptors. Curr Opin Immunol. 1998; 10(3):271-278. (Biology). View Reference
  5. Prlic M, Bevan MJ. Exploring regulatory mechanisms of CD8+ T cell contraction. Proc Natl Acad Sci U S A. 2008; 105(43):16689-16694. (Clone-specific: Flow cytometry). View Reference
  6. Suni MA, Maino VC. Flow cytometric analysis of cell signaling proteins. Methods Mol Biol. 2011; 717:155-169. (Clone-specific: Flow cytometry). View Reference
  7. 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
  8. 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
View All (8) View Less
568146 Rev. 2

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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.

Refer to manufacturer's instructions for use and related User Manuals and Technical Data Sheets before using this product 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.