PE Mouse anti-Btk (pY223)/Itk (pY180)

BD Phosflow™ PE Mouse anti-Btk (pY223)/Itk (pY180)

Name: PE Mouse anti-Btk (pY223)/Itk (pY180)
Brand: BD Phosflow™
SKU: 562753

クローン N35-86

(RUO)

Analyses of BTK (pY223)/ ITK(pY180) expression by Human and Mouse Cells. Human Cells Panel 1a: Flow cytometric analysis of BTK (pY223) expressed by human Ramos cells. Serum-starved Ramos cells (Human Burkitt's lymphoma cell line) were either not stimulated (dashed line histogram) or stimulated (solid line histogram) with Goat F(ab')2 Anti-Human IgM (Southern Biotech, Cat. No. 2022-14) at 37°C for 2 minutes. Cells were fixed in BD Cytofix™ Buffer (Cat. No. 554655; 37°C for 10 min) and permeabilized in BD Phosflow™ Perm Buffer III (Cat. No. 558050) on ice (30 min). Cells were then stained with BD Phosflow™ PE Mouse Anti-Btk (pY223)/Itk (pY180) (Cat. No. 562753). Histograms showing BTK (pY223) expression were generated for gated events with the forward and side-light scatter characteristics of intact cells using a BD FACSCanto™ II Flow Cytometer System. Panel 1b: Western blot analysis of BTK (pY223) expressed by Ramos cells. Lysates from 1 million unstimulated (C) and Anti-IgM stimulated (T) Ramos cells were blotted using Purified Btk (pY223)/Itk (pY180) antibody (0.125, 0.063, and 0.032 μg/ml for Lanes 1, 2, and 3, respectively), HRP Goat Anti-Mouse Ig (Cat. No. 554002) and a chemiluminescent detection system. BTK (pY223) was identified as band of ~77 kDa. Mouse Cells Panel 2a: Flow cytometric analysis of BTK (pY223) expressed by mouse A20 cells. A20 cells (mouse B lymphoma cell line) were either not stimulated (dashed line histogram) or were stimulated (solid line histogram) with Rabbit Anti-Mouse IgG (H+L) (Jackson, Cat. No. 315-005-003) at 37˚C for 2 minutes. The cells were fixed, permed, stained and analyzed by flow cytometry as described above. Panel 2b: Western blot analysis of BTK (pY223) expressed by mouse A20 cells. Lysates from 1 million untimulated (C) and Anti-IgM stimulated (T) A20 cells were blotted as described above with Purified Mouse Anti-Btk (pY223)/Itk (pY180) antibody at 0.032 µg/ml. Lower MW band at 42 kDa represents actin.

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製品詳細

Brand: BD Phosflow™

Alternative Name: Btk; Bruton tyrosine kinase; AGMX1; AT; ATK; BPK; IMD1; PSCTK1; XLA; Itk

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

Isotype: Mouse IgG1, κ

Immunogen: Phosphorylated Human BTK Peptide

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

Vol. Per Test: 5 µl

RRID: AB_2737769

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

Regulatory Status: RUO

Preparation and Storage

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. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.

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.
For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
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.

関連製品

Stain Buffer (FBS) RUO

サイズ 500 mL カタログ番号 554656

Lyse/Fix Buffer 5X RUO

サイズ 250 mL カタログ番号 558049

Perm Buffer III RUO

サイズ 125 mL カタログ番号 558050

Perm/Wash Buffer I RUO

サイズ 125 mL カタログ番号 557885

Fixation Buffer RUO

サイズ 100 mL カタログ番号 554655

Perm Buffer II RUO

サイズ 125 mL カタログ番号 558052

詳細を表示

562753 Rev. 1

抗体の詳細

N35-86

The N35-86 reacts with human and mouse Bruton's tyrosine kinase that is phosphorylated at the tyrosine 223 position, Btk (pY223). Btk is also known as agammaglobulinaemia tyrosine kinase (ATK) and B-cell progenitor kinase (BPK). Btk is a nonreceptor tyrosine kinase whose function is critical for proper B cell development and signaling. It is a member of the Tec family of kinases which includes Tec and Itk. In addition to an N-terminal pleckstrin homology (PH) domain, the Tec proteins contain Src homology domains 2 and 3 (SH2 and SH3) and a stretch of 60-80 amino acids between the PH and SH3 domains termed the Tec homology domain. The activity of Btk is regulated by Src-mediated phosphorylation of the kinase domain at tyrosine 551. This event induces Btk kinase activity and subsequent autophosphorylation at tyrosine 223 in the SH3 domain. Phosphorylated Btk then associates with the cell membrane via the interaction of the PH domain with phosphatidylinositol 3, 4, 5-triphosphate. The PH domain is essential for proper activation and function of Btk. A mutation in the PH domain results in Xid, murine X-linked immunodeficiency, and human X-linked agammaglobulinemia. The orthologous phosphorylation site for rat BTK is Y224. Crossreactivity with human Itk (pY180) was confirmed by immunoprecipitation and Western blot analyses using the N35-86 antibody.

562753 Rev. 1

フォーマットの詳細

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.

フォーマット: PE

励起源: Yellow-Green 488 nm, 532 nm, 561 nm

最大励起波長: 496 nm, 566 nm

最大蛍光波長: 576 nm

562753 Rev.1

引用＆参考文献

Development References (4)

Desiderio S. Role of Btk in B cell development and signaling. Curr Opin Immunol. 9(4):534-540. (Biology). View Reference
Mahajan S, Fargnoli J, Burkhardt AL, Kut SA, Saouaf SJ, Bolen JB. Src family protein tyrosine kinases induce autoactivation of Bruton's tyrosine kinase. Mol Cell Biol. 1995; 15:5304-5311. (Biology). View Reference
Marshall AJ, Niito H, Yun TJ, Clark EA. Regulation of B-cell activation and differentiation by the phosphatidylinositol 3-kinase and phospholipase Cγ pathway. Immunol Rev. 2000; 176:30-46. (Biology). View Reference
Rawlings DJ, Scharenberg AM, Park H, et al. Activation of BTK by a phosphorylation mechanism initiated by SRC family kinases. Science. 1996; 271:822-825. (Biology). View Reference

すべて表示する (4)

562753 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.