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 10 minutes, and then stained with PE Mouse anti-BLNK (pY84) (Cat. No. 558442; both panels), APC Mouse Anti-Human CD3 (Cat. No. 555335, right panel), and PerCP-Cy™5.5 Mouse Anti-Human CD20 (Cat. No. 558021, right 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.
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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 10 minutes, and then stained with PE Mouse anti-BLNK (pY84) (Cat. No. 558442; both panels), APC Mouse Anti-Human CD3 (Cat. No. 555335, right panel), and PerCP-Cy™5.5 Mouse Anti-Human CD20 (Cat. No. 558021, right 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_647182
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 with R-PE under optimum conditions, and unconjugated antibody and free PE 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.
- 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.
- Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
- Cy is a trademark of GE Healthcare.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
Fixation Buffer RUO
Size
100 mL
Cat No.
554655
Perm/Wash Buffer I RUO
Size
125 mL
Cat No.
557885
Perm Buffer II RUO
Size
125 mL
Cat No.
558052
Perm Buffer III RUO
Size
125 mL
Cat No.
558050
APC Mouse Anti-Human CD3 RUO
Size
100 Tests
Cat No.
555335
PerCP-Cy™5.5 Mouse Anti-Human CD20 RUO
Size
50 Tests
Cat No.
558021
558442 Rev. 4
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.
558442 Rev. 4
Format Details
PE
R-Phycoerythrin (PE), is part of the BD family of Phycobiliprotein dyes. This fluorochrome is a multimeric fluorescent phycobiliprotein with excitation maximum (Ex Max) of 496 nm and 566 nm and an emission maximum (Em Max) at 576 nm. PE is designed to be excited by the Blue (488 nm), Green (532 nm) and Yellow-Green (561 nm) lasers and detected using an optical filter centered near 575 nm (e.g., a 575/26-nm bandpass filter). As PE is excited by multiple lasers, this can result in cross-laser excitation and fluorescence spillover on instruments with various combinations of Blue, Green, and Yellow-Green lasers. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
PE
Yellow-Green 488 nm, 532 nm, 561 nm
496 nm, 566 nm
576 nm
558442 Rev.4
Citations & References
Development References (5)
-
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).
-
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
-
Janssen E, Zhang W. Adaptor proteins in lymphocyte activation. Curr Opin Immunol. 2003; 15:269-276. (Biology). View Reference
-
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
-
Wu JN, Koretzky GA. The SLP-76 family of adapter proteins. Semin Immunol. 2004; 16:379-393. (Biology). View Reference
558442 Rev. 4
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.
