Phospho-BTK (Tyr551) Antibody [D4E1]

Application: Reactivity: Human

Usage Information

Dilution
WB1:1000 IF1:500

Application
WB, IF

Reactivity
Human

Source
Rabbit Monoclonal Antibody

Storage Buffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

Predicted MW Observed MW
76 kDa 75 kDa
^*Why do the predicted and actual molecular weights differ? The following reasons may explain differences between the predicted and actual protein molecular weight. Post-translational modifications(e.g., phosphorylation, glycosylation); Splice variants and isoforms; Relative charge; Multimerization.

Datasheet & SDS

Biological Description

Specificity
Phospho-BTK (Tyr551) Antibody [D4E1] detects endogenous levels of total BTK protein only when it is phosphorylated at Tyr551.

Clone
D4E1

Synonym(s)
AGMX1, ATK, BPK, BTK, Tyrosine-protein kinase BTK, Agammaglobulinemia tyrosine kinase, B-cell progenitor kinase, Bruton tyrosine kinase

Background
Phospho‑BTK (Tyr551) denotes Bruton's tyrosine kinase phosphorylated on the activation‑loop tyrosine within its C‑terminal kinase domain, a modification that marks catalytic activation downstream of antigen and innate immune receptors and positions BTK as a central signaling node in B cells and other hematopoietic lineages. BTK belongs to the Tec family and carries an N‑terminal pleckstrin homology domain that binds phosphatidylinositol‑3,4,5‑trisphosphate, followed by a Tec homology region, SH3 and SH2 domains that mediate protein–protein interactions, and a C‑terminal kinase lobe whose activation loop contains Tyr551 as the key regulatory residue. Engagement of the B‑cell receptor or other immunoreceptors leads to PI3K‑dependent generation of PIP3, membrane recruitment of BTK via its PH domain, and exposure of Tyr551 to Src‑family kinase Lyn and Syk, which transphosphorylate Tyr551 and thereby induce the conformational rearrangement characteristic of active tyrosine kinases. Phosphorylation at Tyr551 increases BTK catalytic activity and permits efficient autophosphorylation of Tyr223 in the SH3 domain, stabilizing an open, fully active enzyme configuration that propagates B‑cell receptor signaling by phosphorylating substrates such as PLCγ2 and contributing to downstream Ca²⁺ mobilization, PKC activation, NF‑κB and MAPK pathway engagement, and transcriptional programs for B‑cell activation, survival, and differentiation. Mutation of Tyr551 abolishes BCR‑induced calcium flux while mutation of Tyr223 does not, indicating that Tyr551 phosphorylation is the primary determinant for functional BCR signaling, and biochemical analyses show that activation‑loop phosphorylation directly tunes kinase kinetics and substrate recognition. Phospho‑specific antibodies that recognize BTK only when Tyr551 is phosphorylated demonstrate that Tyr551 phosphorylation increases rapidly upon BCR cross‑linking or TLR4 stimulation and correlates with membrane‑localized, signaling‑competent BTK, while PKCβ‑mediated phosphorylation of Ser180 reduces PH‑domain‑dependent membrane recruitment and thereby limits Tyr551 transphosphorylation, setting a negative feedback threshold for BTK activation. BTK activity regulated at Tyr551 is essential for normal B‑cell development; germline loss‑of‑function of BTK causes X‑linked agammaglobulinemia with a block in pre‑B‑cell maturation, and aberrant or constitutive BTK activation contributes to survival and proliferation of malignant B cells in disorders such as chronic lymphocytic leukemia and mantle cell lymphoma, where Tyr551 phosphorylation provides a mechanistic readout of pathway activation and of pharmacodynamic response to covalent and non‑covalent BTK inhibitors.

Background

Phospho‑BTK (Tyr551) denotes Bruton's tyrosine kinase phosphorylated on the activation‑loop tyrosine within its C‑terminal kinase domain, a modification that marks catalytic activation downstream of antigen and innate immune receptors and positions BTK as a central signaling node in B cells and other hematopoietic lineages. BTK belongs to the Tec family and carries an N‑terminal pleckstrin homology domain that binds phosphatidylinositol‑3,4,5‑trisphosphate, followed by a Tec homology region, SH3 and SH2 domains that mediate protein–protein interactions, and a C‑terminal kinase lobe whose activation loop contains Tyr551 as the key regulatory residue. Engagement of the B‑cell receptor or other immunoreceptors leads to PI3K‑dependent generation of PIP3, membrane recruitment of BTK via its PH domain, and exposure of Tyr551 to Src‑family kinase Lyn and Syk, which transphosphorylate Tyr551 and thereby induce the conformational rearrangement characteristic of active tyrosine kinases. Phosphorylation at Tyr551 increases BTK catalytic activity and permits efficient autophosphorylation of Tyr223 in the SH3 domain, stabilizing an open, fully active enzyme configuration that propagates B‑cell receptor signaling by phosphorylating substrates such as PLCγ2 and contributing to downstream Ca²⁺ mobilization, PKC activation, NF‑κB and MAPK pathway engagement, and transcriptional programs for B‑cell activation, survival, and differentiation. Mutation of Tyr551 abolishes BCR‑induced calcium flux while mutation of Tyr223 does not, indicating that Tyr551 phosphorylation is the primary determinant for functional BCR signaling, and biochemical analyses show that activation‑loop phosphorylation directly tunes kinase kinetics and substrate recognition. Phospho‑specific antibodies that recognize BTK only when Tyr551 is phosphorylated demonstrate that Tyr551 phosphorylation increases rapidly upon BCR cross‑linking or TLR4 stimulation and correlates with membrane‑localized, signaling‑competent BTK, while PKCβ‑mediated phosphorylation of Ser180 reduces PH‑domain‑dependent membrane recruitment and thereby limits Tyr551 transphosphorylation, setting a negative feedback threshold for BTK activation. BTK activity regulated at Tyr551 is essential for normal B‑cell development; germline loss‑of‑function of BTK causes X‑linked agammaglobulinemia with a block in pre‑B‑cell maturation, and aberrant or constitutive BTK activation contributes to survival and proliferation of malignant B cells in disorders such as chronic lymphocytic leukemia and mantle cell lymphoma, where Tyr551 phosphorylation provides a mechanistic readout of pathway activation and of pharmacodynamic response to covalent and non‑covalent BTK inhibitors.

References
https://pubmed.ncbi.nlm.nih.gov/9188445/ https://pubmed.ncbi.nlm.nih.gov/19206206/

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%