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PathScan® Phospho-Btk (Tyr223) Sandwich ELISA kit #23843

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ELISA Image 1: PathScan® Phospho-Btk (Tyr223) Sandwich ELISA kit
Figure 1. Treatment of Ramos cells with anti-human IgM stimulates phosphorylation of Btk at Tyr223, detected by PathScan® Phospho-Btk (Tyr223) Sandwich ELISA Kit #23843, but does not affect the level of total Btk, detected by PathScan® Total Btk Sandwich ELISA Kit #24547. The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blots using Btk (D3H5) Rabbit mAb #8547 (left panel) or Phospho-Btk (Tyr223) (D9T6H) Rabbit mAb #87141 (right panel) are shown in the bottom figure.
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To Purchase # 23843**

Important Ordering Details

Custom Ordering Details:

If kit quantities from the same lot are needed in unlisted sizes, contact us for processing time and pricing.

Looking for this ELISA kit in a 384-well format? Inquire for availability, processing time, and pricing.

Supporting Data

REACTIVITY H
Application Key:
  • ELISA-ELISA 
Species Cross-Reactivity Key:
  • H-Human 
  • Product Includes
  • Related Products
Product IncludesVolumeSolution Color
Btk Mouse mAb Coated Microwells #6097696 tests
P-Btk (Tyr223) Rabbit Detection mAb #882361 eaGreen (Lyophilized)
Anti-rabbit IgG, HRP-linked Antibody (ELISA Formulated) #132721 eaRed (Lyophilized)
Detection Antibody Diluent #1333911 mlGreen
HRP Diluent #1351511 mlRed
TMB Substrate #700411 ml
STOP Solution #700211 ml
Sealing Tape #545032 ea
ELISA Wash Buffer (20X) #980125 ml
ELISA Sample Diluent #1108325 mlBlue
Cell Lysis Buffer (10X) #980315 ml

Kit contents scale proportionally with size, except sealing tape.
Example: The V1 kit contains 5X the listed quantities above, but will exclude the sealing tape.

The microwell plate is supplied as 12 8-well modules - Each module is designed to break apart for 8 tests.

Product Information

Product Description

The PathScan® Phospho-Btk (Tyr223) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Btk protein phosphorylated at Tyr223. A Btk mouse antibody has been coated onto the microwells. After incubation with cell lysates, both phospho- and non-phospho-Btk proteins are captured by the coated antibody. Following extensive washing, a phospho-Btk (Tyr223) rabbit antibody is added to detect the captured phospho-Btk protein. Anti-rabbit IgG, HRP-linked Antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of Btk phosphorylated at Tyr223.

*Antibodies in this kit are custom formulations specific to kit.

Protocol

Specificity / Sensitivity

PathScan® Phospho-Btk (Tyr223) Sandwich ELISA Kit detects endogenous levels of Btk protein phosphorylated at Tyr223 in human cells. The kit sensitivity is shown in Figure 1. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

Species Reactivity:

Human

Background

Bruton's tyrosine kinase (Btk) is a member of the Btk/Tec family of cytoplasmic tyrosine kinases. Like other Btk family members, it contains a pleckstrin homology (PH) domain and Src homology SH3 and SH2 domains. Btk plays an important role in B cell development (1,2). Activation of B cells by various ligands is accompanied by Btk membrane translocation mediated by its PH domain binding to phosphatidylinositol-3,4,5-trisphosphate (3-5). The membrane-localized Btk is active and associated with transient phosphorylation of two tyrosine residues, Tyr551 and Tyr223. Tyr551 in the activation loop is transphosphorylated by the Src family tyrosine kinases, leading to autophosphorylation at Tyr223 within the SH3 domain, which is necessary for full activation (6,7). The activation of Btk is negatively regulated by PKCβ through phosphorylation of Btk at Ser180, which results in reduced membrane recruitment, transphosphorylation, and subsequent activation (8). The PKC inhibitory signal is likely to be a key determinant of the B cell receptor signaling threshold to maintain optimal Btk activity (8).

Pathways

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Database Links

UniProt ID: Q06187


Entrez-Gene Id: 695


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