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Alexa Fluor® 647 Mouse Anti-Stat5 (pY694)
Alexa Fluor® 647 Mouse Anti-Stat5 (pY694)

Flow cytometric analysis of Stat5 (pY694). TF-1 cells (Human erythroleukemia; ATCC CRL-2003), a GM-CSF dependent cell line, were starved overnight in IMDM with 0.1% FBS without GM-CSF. The following day, cells were either left unstimulated (unshaded) or stimulated (shaded) with 25 ng/ml GM-CSF recombinant protein (Cat. No. 550068) for 15 minutes at 37°C. Cells were fixed with BD Cytofix™ buffer (10 minutes at 37°C; Cat. No. 554655) and then permeabilized by adding BD Phosflow™ Perm Buffer III (30 minutes on ice; Cat. No. 558050). Cells were then washed twice in BD Pharmingen™ Stain Buffer (Cat. No. 554656), and stained with the Alexa Fluor® 647 Mouse Anti-Stat5 (pY694) antibody. The cells were analyzed on a BD FACSCalibur™ flow cytometer.  For intracellular staining of human whole blood, BD Phosflow™ Lyse/Fix buffer (MN 558049) may be used for fixation.

Flow cytometric analysis of Stat5 (pY694). TF-1 cells (Human erythroleukemia; ATCC CRL-2003), a GM-CSF dependent cell line, were starved overnight in IMDM with 0.1% FBS without GM-CSF. The following day, cells were either left unstimulated (unshaded) or stimulated (shaded) with 25 ng/ml GM-CSF recombinant protein (Cat. No. 550068) for 15 minutes at 37°C. Cells were fixed with BD Cytofix™ buffer (10 minutes at 37°C; Cat. No. 554655) and then permeabilized by adding BD Phosflow™ Perm Buffer III (30 minutes on ice; Cat. No. 558050). Cells were then washed twice in BD Pharmingen™ Stain Buffer (Cat. No. 554656), and stained with the Alexa Fluor® 647 Mouse Anti-Stat5 (pY694) antibody. The cells were analyzed on a BD FACSCalibur™ flow cytometer.  For intracellular staining of human whole blood, BD Phosflow™ Lyse/Fix buffer (MN 558049) may be used for fixation.

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_399882
Aqueous buffered solution containing BSA 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 to Alexa Fluor® 647 under optimum conditions, and unreacted Alexa Fluor® 647 was 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 purified or conjugated 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

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. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  3. 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.
  4. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
  5. Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
  6. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  7. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  8. 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.
612599 Rev. 7
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.

612599 Rev. 7
Format Details
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Alexa Fluor™ 647
Alexa Fluor™ 647 Dye is part of the BD red family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 653-nm and an emission maximum (Em Max) at 669-nm. Alexa Fluor 647 is designed to be excited by the Red laser (627-640 nm) and detected using an optical filter centered near 520-nm (e.g., a 660/20 nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
Alexa Fluor™ 647
Red 627-640 nm
653 nm
669 nm
612599 Rev.7
Citations & References
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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. Imada K, Leonard WJ. The Jak-STAT pathway. Mol Immunol. 2000; 37:1-11. (Biology). View Reference
  4. 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
  5. Liu KD, Gaffen SL, Goldsmith MA. JAK/STAT signaling by cytokine receptors. Curr Opin Immunol. 1998; 10(3):271-278. (Biology). View Reference
  6. 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
  7. Suni MA, Maino VC. Flow cytometric analysis of cell signaling proteins. Methods Mol Biol. 2011; 717:155-169. (Clone-specific: Flow cytometry). 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
612599 Rev. 7

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.

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.