Human Phospho-STAT5a/b (Y694/Y699) Antibody

(3 citations)   
  • Specificity
    Detects human STAT5a/b when phosphorylated at Y694/Y699 in Western blots.
  • Source
    Polyclonal Rabbit IgG
  • Purification
    Antigen Affinity-purified
  • Immunogen
    Synthetic phosphopeptide containing human STAT5b Y699 site (aa sequence of this peptide is identical to a correspondent region of human STAT5a containing Y694).
  • Formulation
    Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied as a 0.2 µm filtered solution in PBS.
  • Label
    Unconjugated
Applications
  •  
    Recommended
    Concentration
    Sample
  • Western Blot
    1 µg/mL
    See below
  • Simple Western
    10 µg/mL
    See below
  • Immunocytochemistry
    5-15 µg/mL
    See below
  • Intracellular Staining by Flow Cytometry
    2.5 µg/106 cells
    See below
Please Note: Optimal dilutions should be determined by each laboratory for each application. General Protocols are available in the Technical Information section on our website.
Data Examples
Detection of Human Phospho-STAT5a/b (Y694/Y699) by Western Blot. Western blot shows lysates of Daudi human Burkitt's lymphoma cell line and U937 human histiocytic lymphoma cell line untreated (-) or treated (+) with 500 U/mL Recombinant Human
IFN‑ alpha  2a (Catalog # 11100-1) for 15 minutes. Gels were loaded with 20 μg of cytoplasmic (Cyto) and 10 μg of nuclear extracts (Nuc). PVDF membrane was probed with 1 µg/mL Rabbit Anti-Human Phospho-STAT5a/b (Y694/Y699) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF4190) followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # HAF008). A specific band for Phospho-STAT5a/b (Y694/Y699) was detected at approximately 105 kDa (as indicated). For additional reference, duplicate lysates were probed with 1.0  µg/mL Human/Mouse STAT5a/b Pan Specific Antigen Affinity-purified Polyclonal Antibody (lower panel, Catalog # AF2168). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.
Detection of Human Phospho-STAT5a/b (Y694/Y699) by Simple WesternTM.

Simple Western lane view shows lysates of Daudi human Burkitt's lymphoma cell line untreated (-) or treated (+) with 500 U/mL Recombinant Human IFN‑ alpha A (Catalog # 11100-1) for 15 minutes, loaded at 0.2 mg/mL. A specific band was detected for STAT5a/b at approximately 68 kDa (as indicated) using 10 µg/mL of Rabbit Anti-Human Phospho-STAT5a/b (Y694/Y699) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF4190). This experiment was conducted under reducing conditions and using the
12-230 kDa separation system.

*Non-specific interaction with the 230 kDa Simple Western standard may be seen with this antibody.

Intracellular Staining by Flow Cytometry
Detection of STAT5a/b in Daudi Human Cell Line by Flow Cytometry. Daudi human Burkitt's lymphoma cell line was unstimulated (light orange filled histogram) or treated with 500 U/mL Recombinant Human IFN-alpha (Catalog # 11100-1) for 20 minutes, then stained with Rabbit Anti-Human Phospho-STAT5a/b (Y694/Y699) Antigen Affinity‑purified Polyclonal Antibody (Catalog # AF4190, dark orange filled histogram) or isotype control antibody (Catalog # AB‑105‑C, open histogram), followed by Allophycocyanin-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # F0111). To facilitate intracellular staining, cells were fixed with paraformaldehyde and permeabilized with ice-cold methanol.
Immunocytochemistry
STAT5a/b in Daudi Human Burkitt's Lymphoma Cells. STAT5a/b phosphorylated at Y694/Y699 was detected in immersion fixed IFN-alpha treated Daudi human Burkitt's lymphoma cell line using 10 µg/mL Rabbit Anti-Human Phospho-STAT5a/b (Y694/Y699) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF4190) for 3 hours at room temperature. Cells were stained with the NorthernLights™ 557-conjugated Anti-Rabbit IgG Secondary Antibody (red, upper panel; Catalog # NL004) and counterstained with DAPI (blue, lower panel). View our protocol for Fluorescent ICC Staining of Cells on Coverslips.
Preparation and Storage
  • Reconstitution
    Reconstitute at 0.2 mg/mL in sterile PBS.
  • Shipping
    The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below. *Small pack size (SP) is shipped with polar packs. Upon receipt, store it immediately at -20 to -70 °C
  • Stability & Storage
    Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
    • 12 months from date of receipt, -20 to -70 °C as supplied.
    • 1 month, 2 to 8 °C under sterile conditions after reconstitution.
    • 6 months, -20 to -70 °C under sterile conditions after reconstitution.
Background: STAT5a/b

Signal transduction and activator of transcription 5 (STAT5) is a member of the Jak/STAT signal transduction pathway and is activated by a variety of cytokines (IL‑22, IL-6, IFN-alpha ). STAT5 has two isoforms (A and B) that share 93% amino acid identity and bind the DNA consensus site TTCN3GAA. STAT5 mediates cytokine signaling by acting as a signal transducer in the cytoplasm and, upon phosphorylation, translocates to the nucleus and activates transcription of specific genes. STAT5 is involved in a wide array of biological processes ranging from regulating apoptosis to adult mammary gland proliferation, differentiation, and survival.

  • Long Name:
    Signal Transducer and Activator of Transcription 5b
  • Alternate Names:
    MGF; STAT5; STAT5A signal transducer and activator of transcription 5A
Related Research Areas
Citations:

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

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Species
Applications
Sample Type
  1. Colocalization of the VEGF-R2 and the common IL-3/GM-CSF receptor beta chain to lipid rafts leads to enhanced p38 activation.
    Authors: Saulle E, Riccioni R, Coppola S, Parolini I, Diverio D, Riti V, Mariani G, Laufer S, Sargiacomo M, Testa U
    Br. J. Haematol., 2009;145(3):399-411.
    Species: Human
    Sample Type: Cell Lysates
    Application: Western Blot,
  2. MiR-21 is under control of STAT5 but is dispensable for mammary development and lactation.
    Authors: Feuermann, Yonatan, Kang, Keunsoo, Shamay, Avi, Robinson, Gertraud, Hennighausen, Lothar
    PLoS ONE, 2014;9(1):e85123.
    Species: Mouse
    Sample Type: Cell Lysates
    Application: ChIP,
  3. Erythropoietin production in neuroepithelial and neural crest cells during primitive erythropoiesis.
    Authors: Suzuki N, Hirano I, Pan X, Minegishi N, Yamamoto M
    Nat Commun, 2013;4(0):2902.
    Species: Mouse
    Sample Type: Whole Cells
    Application: ELISA Capture,
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