Alexa Fluor® 647 Mouse Anti-Stat1 (pY701)

BD Phosflow™ Alexa Fluor® 647 Mouse Anti-Stat1 (pY701)

Name: Alexa Fluor® 647 Mouse Anti-Stat1 (pY701)
Brand: BD Phosflow™
SKU: 562070

Clone 4a

(RUO)

Flow cytometric analysis of Stat1 (pY701). U-937 cells (Human histiocytic lymphoma; ATCC CRL-1593.2) were either left unstimulated (unshaded) or stimulated (shaded) with 1000 U/mL recombinant human IFN-γ (Cat. No. 554617) for 15 minutes at 37°C. Cells were fixed with BD Cytofix™ buffer (Cat. No. 554655) for 10 minutes at 37°C and then permeabilized by adding BD Phosflow™ Perm Buffer III (Cat. No. 558050 for 30 minutes on ice. Cells were then washed twice in BD Pharmingen™ Stain Buffer (Cat. No. 554656) and stained with the Alexa Fluor® 647 mouse anti-Stat1 (pY701) antibody. Cells were analyzed on a BD FACSCalibur™ flow cytometery instrument. For intracellular staining of human whole blood, BD Phosflow™ Lyse/Fix buffer (Cat. No. 558049) may be used for fixation.

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Product Details

Brand: BD Phosflow™

Reactivity: Human (QC Testing), Mouse (Tested in Development)

Isotype: Mouse IgG2a

Immunogen: Phosphorylated Human Stat1 Peptide

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

Vol. Per Test: 5 µl

RRID: AB_10896129

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 to Alexa Fluor® 647 under optimum conditions, and unreacted Alexa Fluor® 647 was removed.

Recommended Assay Procedures

For more information about BD Phosflow™: Please refer to http://www.bdbiosciences.com/support/resources/phosflow/index.jsp

Investigators may also find the following protocols to be helpful:

Phosflow protocol for human PBMC: Please refer to http://www.bdbiosciences.com/documents/Phosflow_Protocol_for_Human_PBMCs.pdf

Phosflow protocol for human whole blood: Please refer to http://www.bdbiosciences.com/documents/Phosflow_Protocol_for_Human_Whole_Blood_Samples.pdf

Phosflow protocol for adherent cells: Please refer to http://www.bdbiosciences.com/support/resources/protocols/protocol_adherent.jsp

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

Companion Products

Stain Buffer (FBS) RUO

Size 500 mL Cat No. 554656

Lyse/Fix Buffer 5X RUO

Size 250 mL Cat No. 558049

Fixation Buffer RUO

Size 100 mL Cat No. 554655

Perm Buffer III RUO

Size 125 mL Cat No. 558050

562070 Rev. 1

Antibody Details

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 as well as subsequent recruitment and 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. Stat1 and Stat2 are components of the ISGF3 (Interferon-Stimulated Gene Factor 3) complex, the primary transcription activator induced by interferon binding to a specific cell-surface receptor. Stat1 has two alternatively spliced isoforms, 91-kDa Stat1α and 84-kDa Stat1β; Stat1α has 38 additional C-terminal amino acids. In response to the binding of IFNα, IFNγ, EGF, PDGF, or CSF-1 to their respective receptors, the Stat1 subunits become tyrosine-phosphorylated at Y701, and the complex translocates to the nucleus. This forms an active complex that includes the DNA-binding p48 subunit, and is responsible for modulating interferon-stimulated genes (ISGs) transciption.

The 4a monoclonal antibody recognizes the phosphorylated Y701 in Stat1α and Stat1β.

562070 Rev. 1

Format Details

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.

Format: Alexa Fluor™ 647

Excitation Source: Red 627-640 nm

Excitation Max: 653 nm

Emission Max: 669 nm

562070 Rev.1

Citations & References

View product citations for antibody "562070" on CiteAb

Development References (3)

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
Darnell JE Jr. STATs and gene regulation. Science. 1997; 277(5332):1630-1635. (Biology). View Reference
Fu XY, Zhang JJ. Transcription factor p91 interacts with the epidermal growth factor receptor and mediates activation of the c-fos gene promoter. Cell. 1993; 74(6):1135-1145. (Biology). View Reference

562070 Rev. 1

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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.