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RB744 Mouse Anti-Stat5 (pY694)
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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 (Cat. No. 560760) were reconstituted in BD Pharmingen™ Stain Buffer (Cat. No. 554656) for 15 minutes at room temperature. These cells are fixed, permeabilized, and lyophilized PBMC that are either untreated (Dashed Line) or treated (Solid Line) 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 Horizon™ BUV395 Mouse Anti-Human CD4 antibody (Cat. No. 564724) and BD Phosflow™ RB744 Mouse Anti-Human Stat5 (pY694) antibody (Cat. No. 570506/570507). 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 FACSymphony™ A5 SE Cell Analyzer System and FlowJo™ Software.

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Product Details
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BD Phosflow™
Signal transducer and activator of transcription 5; MGF; MPF
Human (QC Testing), Mouse, Rhesus, Baboon (Reported), Rat,Sheep (Predicted)
Mouse IgG1, κ
Phosphorylated Human Phosphorylated Stat5 Peptide
Intracellular staining (flow cytometry) (Routinely Tested)
5 µl/test
6776, 6777, 20850
AB_3685799
Aqueous buffered solution containing ≤0.09% sodium azide.
RUO


Preparation And Storage

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. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.

Product Notices

  1. When using high concentrations of antibody, background binding of this dye to erythroid fragments produced by ammonium chloride-based lysis, such as with BD Pharm Lyse™ Lysing Buffer (Cat. No. 555899), has been observed when the antibody conjugate was present during the lysis procedure. This may cause nonspecific staining of target cells, such as leukocytes, which have bound the resulting erythroid fragments. This background can be mitigated by any of the following: titrating the antibody conjugate to a lower concentration, fixing samples with formaldehyde, or removing erythrocytes before staining (eg, gradient centrifugation or pre-lysis with wash). This background has not been observed when cells were lysed with BD FACS™ Lysing Solution (Cat. No. 349202) after staining.
  2. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  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. Please observe the following precautions: We recommend that special precautions be taken (such as wrapping vials, tubes, or racks in aluminum foil) to protect exposure of conjugated reagents, including cells stained with those reagents, to any room illumination. Absorption of visible light can significantly affect the emission spectra and quantum yield of tandem fluorochrome conjugates.
  6. 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.
  7. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  8. 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.
  9. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
  10. For U.S. patents that may apply, see bd.com/patents.

Data Sheets

570507 Rev. 1
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.

570507 Rev. 1
Format Details
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RB744
The BD Horizon RealBlue™ 744 (RB744) Dye is part of the BD® family of blue dyes. It is a tandem fluorochrome with an excitation maximum (Ex Max) at 498-nm and an emission maximum (Em Max) at 746-nm as measured using an antibody-dye conjugate. Driven by BD® innovation, RB744 can be used on both spectral and conventional cytometers and is designed to be excited by the Blue laser (488-nm) with minimal excitation by the 561-nm Yellow-Green laser. For conventional instruments equipped with a Blue laser (488-nm), we recommend using an optical filter centered near 750-nm (e.g., a 750/60-nm bandpass filter).
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RB744
498 nm
746 nm
570507 Rev.1
Citations & References
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Development References (10)

  1. Belarif L, Mary C, Jacquemont L, et al. IL-7 receptor blockade blunts antigen-specific memory T cell responses and chronic inflammation in primates.. Nat Commun. 2018; 9(1):4483. (Clone-specific: Intracellular Staining/Flow Cytometry). View Reference
  2. DeGottardi MQ, Okoye AA, Vaidya M, et al. Effect of Anti-IL-15 Administration on T Cell and NK Cell Homeostasis in Rhesus Macaques.. J Immunol. 2016; 197(4):1183-98. (Clone-specific: Intracellular Staining/Flow Cytometry). 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: Intracellular Staining/Flow Cytometry). View Reference
  4. Krutzik PO, Clutter MR, Nolan GP. Coordinate analysis of murine immune cell surface markers and intracellular phosphoproteins by flow cytometry.. J Immunol. 2005; 175(4):2357-65. (Clone-specific: Intracellular Staining/Flow Cytometry). View Reference
  5. 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: Intracellular Staining/Flow Cytometry). View Reference
  6. Liu KD, Gaffen SL, Goldsmith MA. JAK/STAT signaling by cytokine receptors. Curr Opin Immunol. 1998; 10(3):271-278. (Biology). View Reference
  7. 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: Intracellular Staining/Flow Cytometry). View Reference
  8. Suni MA, Maino VC. Flow cytometric analysis of cell signaling proteins. Methods Mol Biol. 2011; 717:155-169. (Clone-specific: Intracellular Staining/Flow Cytometry). View Reference
  9. Tusi BK, Wolock SL, Weinreb C, et al. Population snapshots predict early haematopoietic and erythroid hierarchies.. Nature. 2018; 555(7694):54-60. (Clone-specific: Intracellular Staining/Flow Cytometry). View Reference
  10. 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
View All (10) View Less
570507 Rev. 1

 

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.

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.