BV421 Mouse Anti-Stat5 (pY694)

BD Phosflow™ BV421 Mouse Anti-Stat5 (pY694)

Name: BV421 Mouse Anti-Stat5 (pY694)
Brand: BD Phosflow™
SKU: 562984

Clone 47/Stat5(pY694)

(RUO)

Flow cytometric analysis of Stat5 (pY694) expression in IL-2-treated human peripheral blood lymphocytes. Human whole blood was either stimulated with 100 ng/ml (final concentration) of BD Pharmingen™ Recombinant Human IL-2 protein (Cat. No. 554903) for 15 minutes at 37ºC (solid line histogram) or was left unstimulated (dashed line histogram). The erythrocytes were lysed and the leukocytes were fixed with 1× BD Phosflow™ Lyse/Fix Buffer (Cat. No. 558049) for 15 minutes at 37ºC. The leukocytes were washed, permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for 30 minutes, washed and then stained with BD Phosflow™ BV421 Mouse Anti-Stat5 (pY694) antibody (Cat. No. 562984). The fluorescence histograms were derived from events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometry was performed using a BD™ LSR II Flow Cytometer System.

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Product Details

Brand: BD Phosflow™

Alternative Name: Signal transducer and activator of transcription 5; MGF; MPF

Reactivity: Human (QC Testing)

Isotype: Mouse IgG1, κ

Immunogen: Phosphorylated Human Phosphorylated Stat5 Peptide

Application: Intracellular staining (flow cytometry) (Routinely Tested)

Vol. Per Test: 5 µl

RRID: AB_2737931

Storage Buffer: Aqueous buffered solution containing BSA and ≤0.09% sodium azide.

Regulatory Status: 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 BD Horizon BV421 under optimum conditions, and unconjugated antibody and free BD Horizon BV421 were removed.

Product Notices

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).
Source of all serum proteins is from USDA inspected abattoirs located in the United States.
Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
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.
For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
Pacific Blue™ is a trademark of Molecular Probes, Inc., Eugene, OR.
Brilliant Violet™ 421 is a trademark of Sirigen.

Companion Products

Stain Buffer (FBS) RUO

Size 500 mL Cat No. 554656

Lyse/Fix Buffer 5X RUO

Size 250 mL Cat No. 558049

Perm Buffer III RUO

Size 125 mL Cat No. 558050

Recombinant Human IL-2 RUO

Size 10 µg Cat No. 554603

562984 Rev. 1

Antibody Details

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.

The antibody was conjugated to BD Horizon™ BV421 which is part of the BD Horizon™ Brilliant Violet™ family of dyes. With an Ex Max of 407-nm and Em Max at 421-nm, BD Horizon™ BV421 can be excited by the violet laser and detected in the standard Pacific Blue™ filter set (eg, 450/50-nm filter). BD Horizon™ BV421 conjugates are very bright, often exhibiting a 10 fold improvement in brightness compared to Pacific Blue™ conjugates.

562984 Rev. 1

Format Details

BV421

The BD Horizon Brilliant Violet™ 421 (BV421) Dye is part of the BD Horizon Brilliant Violet™ family of dyes. This polymer-technology based dye has an excitation maximum (Ex Max) of 407-nm and an emission maximum (Em Max) at 423-nm. Driven by BD innovation, BV421 is designed to be excited by the violet laser (405-nm) and detected using an optical filter centered near 420-nm (e.g., a 431/28-nm or 450/50-nm bandpass filter). BV421 is an ideal alternative for V450 as it is approximately ten times brighter with less spillover into the BV510/V500 detector. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.

Format: BV421

Excitation Source: Violet 405 nm

Excitation Max: 407 nm

Emission Max: 423 nm

562984 Rev.1

Citations & References

Development References (8)

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
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
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
Liu KD, Gaffen SL, Goldsmith MA. JAK/STAT signaling by cytokine receptors. Curr Opin Immunol. 1998; 10(3):271-278. (Biology). View Reference
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
Suni MA, Maino VC. Flow cytometric analysis of cell signaling proteins. Methods Mol Biol. 2011; 717:155-169. (Clone-specific: Flow cytometry). View Reference
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
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)

562984 Rev. 1

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