Analysis of BLNK (pY84) in human Burkitt's lymphoma and peripheral blood lymphocytes. Ramos cells (left panel) and human peripheral blood mononuclear cells (PBMC, right panel) were either stimulated (solid-line histograms) by cross-linking of surface IgM with purified Goat anti-Human IgM (SouthernBiotech) at 37°C for 2 minutes or unstimulated (dashed-line histograms). The cells were fixed with pre-warmed BD Cytofix™ buffer (Cat. No. 554655) for 10 minutes at 37ºC, then permeabilized with BD Phosflow™ Perm/Wash Buffer I (Cat. No. 557885) at room temperature for 30 minutes, and then stained with Alexa Fluor® 647 anti-BLNK (pY84) (both panels), APC anti-human CD3 mAb UCHT1 (Cat. No. 555335, bottom panel), and PerCP-Cy5.5 anti-human CD20 mAb H1(FB1) (Cat. No. 558021, bottom panel). For data analysis of PBMC, CD3-negative CD20-positive lymphocytes were selected by their staining and scatter profiles. Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.
Analysis of BLNK (pY84) in human Burkitt's lymphoma and peripheral blood lymphocytes. Ramos cells (left panel) and human peripheral blood mononuclear cells (PBMC, right panel) were either stimulated (solid-line histograms) by cross-linking of surface IgM with purified Goat anti-Human IgM (SouthernBiotech) at 37°C for 2 minutes or unstimulated (dashed-line histograms). The cells were fixed with pre-warmed BD Cytofix™ buffer (Cat. No. 554655) for 10 minutes at 37ºC, then permeabilized with BD Phosflow™ Perm/Wash Buffer I (Cat. No. 557885) at room temperature for 30 minutes, and then stained with Alexa Fluor® 647 anti-BLNK (pY84) (both panels), APC anti-human CD3 mAb UCHT1 (Cat. No. 555335, bottom panel), and PerCP-Cy5.5 anti-human CD20 mAb H1(FB1) (Cat. No. 558021, bottom panel). For data analysis of PBMC, CD3-negative CD20-positive lymphocytes were selected by their staining and scatter profiles. Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.
Product Details
BD Phosflow™
Human (QC Testing), Mouse (Tested in Development)
Mouse BALB/c IgG2b, κ
Phosphorylated Human BLNK Peptide
Intracellular staining (flow cytometry) (Routinely Tested)
20 µl
AB_647111
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.
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).
- 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.
- Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
- 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.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
558443 Rev. 2
Antibody Details
J117-1278
B cell activation is initiated by crosslinking the B cell receptor, which leads to activation of non-receptor protein tyrosine kinases (PTK), including Btk, Syk, and three Src kinases, Fyn, Lyn, and Blk. Activated PTKs then phosphorylate multiple cellular proteins involved in B lymphocyte signaling. Syk is responsible for the tyrosine phosphorylation of B cell linker protein (BLNK), a member of the SLP-76 family of adapter proteins. Phosphorylation of human BLNK at tyrosines 84, 178, and 189 (Y84, Y178, and Y189) creates docking sites for PLCγ2, leading to the activation of downstream signaling pathways.
The J117-1278 monoclonal antibody recognizes the phosphorylated Y84 of BLNK.
558443 Rev. 2
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.
Alexa Fluor™ 647
Red 627-640 nm
653 nm
669 nm
558443 Rev.2
Citations & References
Development References (5)
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Chiu CW, Dalton M, Ishiai M, Kurosaki T, Chan AC. BLNK: molecular scaffolding through 'cis'-mediated organization of signaling proteins. EMBO J. 2002; 21:6461-6472. (Biology).
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Espeli M, Mancini S, Breton C, Poirier F, Schiff C . Impaired B-cell development at the pre-Bll-cell stage in galectin-1-deficient mice due to inefficient pre-Bll/stromal cell interactions. Blood. 2009; 113(23):5878-5886. (Clone-specific: Flow cytometry). View Reference
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Janssen E, Zhang W. Adaptor proteins in lymphocyte activation. Curr Opin Immunol. 2003; 15:269-276. (Biology). View Reference
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Pighi C, Gu T, Dalai I, et al. Phospho-proteomic analysis of mantle cell lymphoma cells suggests a pro-survival role of B-cell receptor signaling. Cell Oncol. 2011; 34(2):141-153. (Clone-specific: Flow cytometry, Western blot). View Reference
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Wu JN, Koretzky GA. The SLP-76 family of adapter proteins. Semin Immunol. 2004; 16:379-393. (Biology). View Reference
558443 Rev. 2
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.
