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Invitrogen

Phospho-BTK/ITK (Tyr551, Tyr511) Monoclonal Antibody (M4G3LN), PerCP-eFluor™ 710, eBioscience™

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Additional Information:

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Product Details

46-9015-42

Applications
Tested Dilution
Publications

Flow Cytometry (Flow)

5 µL (0.5 µg)/test
-
Product Specifications

Species Reactivity

Human, Mouse

Host/Isotype

Mouse / IgG2b, kappa

Recommended Isotype Control

Mouse IgG2b kappa Isotype Control (eBMG2b), PerCP-eFluor™ 710, eBioscience™

Class

Monoclonal

Type

Antibody

Clone

M4G3LN

Conjugate

PerCP-eFluor™ 710 PerCP-eFluor™ 710 PerCP-eFluor™ 710

Excitation/Emission Max

482/708 nm View spectra spectra

Form

Liquid

Concentration

5 µL/Test

Purification

Affinity chromatography

Storage buffer

PBS, pH 7.2, with BSA

Contains

0.09% sodium azide

Storage conditions

4°C, store in dark, DO NOT FREEZE!

Shipping conditions

Ambient (domestic); Wet ice (international)

RRID

AB_2573864

Product Specific Information

Description: This M4G3LN monoclonal antibody recognizes human and mouse Bruton's tyrosine kinase (also known as BTK, agammaglobulinaemia tyrosine kinase, B-cell progenitor kinase)/interleukin-2-inducible tyrosine kinase (also known as ITK, T-cell-specific kinase, Tyrosine-protein kinase Lyk) when phosphorylated on Y551 and Y511, respectively.

In addition to Src family and SYK family kinases, B cells and T cells express a third family of receptor-proximal kinases called the Tec kinase family. The B cell-specific Tec family kinase is BTK; T cells express ITK as well as Tec and Rlk. Tec family kinases play an indispensable role in activation of phospholipase C and activation of calcium-dependent signaling cascades following antigen receptor triggering. BTK has been implicated in propagating signals downstream of LPS-TLR4 in B cells via signaling through the TLR4 co-receptor RP105/CD180. Mice congenitally lacking BTK or ITK show profound defects in B cell or T cell development and signaling, respectively. People with mutations or deletions in BTK suffer from X-linked agammaglobulinemia and X-linked immunodeficiency.

Specificity of this M4G3LN clone was determined by ELISA, flow cytometry, and western blotting.

Applications Reported: This M4G3LN antibody has been reported for use in intracellular staining followed by flow cytometric analysis.

Applications Tested: This M4G3LN antibody has been pre-titrated and tested by intracellular staining followed by flow cytometric analysis of stimulated normal human peripheral blood cells. This can be used at 5 µL (0.5 µg) per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test.

Staining Protocol: All protocols work well for this monoclonal antibody. Use of Protocol A: Two-step protocol: intracellular (cytoplasmic) proteins allows for the greatest flexibility for detection of surface and intracellular (cytoplasmic) proteins. Use of Protocol B: One-step protocol: intracellular (nuclear) proteins is recommended for staining of transcription factors in conjunction with surface and phosphorylated intracellular (cytoplasmic) proteins. Protocol C: Two-step protocol: Fixation/Methanol allows for the greatest discrimination of phospho-specific signaling between unstimulated and stimulated samples, but with limitations on the ability to stain specific surface proteins (refer to "Clone Performance Following Fixation/Permeabilization" located in the BestProtocols Section under the Resources tab online). All Protocols can be found in the Flow Cytometry Protocols: "Staining Intracellular Antigens for Flow Cytometry Protocol" located in the BestProtocols® Section under the Resources tab online.

PerCP-eFluor® 710 emits at 710 nm and is excited with the blue laser (488 nm); it can be used in place of PerCP-Cyanine5.5. We recommend using a 710/50 bandpass filter, however, the 695/40 bandpass filter is an acceptable alternative. Please make sure that your instrument is capable of detecting this fluorochrome.

Fixation: Samples can be stored in IC Fixation Buffer (Product # 00-8222) (100 µL of cell sample + 100 µL of IC Fixation Buffer) or 1-step Fix/Lyse Solution (Product # 00-5333) for up to 3 days in the dark at 4°C with minimal impact on brightness and FRET efficiency/compensation. Some generalizations regarding fluorophore performance after fixation can be made, but clone specific performance should be determined empirically.

Excitation: 488 nm; Emission: 710 nm; Laser: Blue Laser.

Filtration: 0.2 µm post-manufacturing filtered.

Target Information

In addition to Src family and SYK family kinases, B cells and T cells express a third family of receptor-proximal kinases called the Tec kinase family. The B cell-specific Tec family kinase is BTK; T cells express ITK as well as Tec and Rlk. Tec family kinases play an indispensable role in activation of phospholipase C and activation of calcium-dependent signaling cascades following antigen receptor triggering. BTK has been implicated in propagating signals downstream of LPS-TLR4 in B cells via signaling through the TLR4 co-receptor RP105/CD180. Mice congenitally lacking BTK or ITK show profound defects in B cell or T cell development and signaling, respectively. People with mutations or deletions in BTK suffer from X-linked agammaglobulinemia and X-linked immunodeficiency.

For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.

How to use the Panel Builder

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Bioinformatics

Protein Aliases: agammaglobulinaemia tyrosine kinase; agammaglobulinemia tyrosine kinase; ATK; B-cell progenitor kinase; BPK; Bruton agammaglobulinemia tyrosine kinase; bruton tyrosine kinase; Bruton's tyrosine kinase; dominant-negative kinase-deficient Brutons tyrosine kinase; frameshift mutation results in truncated protein; homolog of mouse T-cell itk/tsk; IL-2-inducible T-cell kinase; interleukin-2-inducible T cell kinase; interleukin-2-inducible T-cell kinase; kinase EMB; kinase EMT; kinase TLK; T-cell-specific kinase; Tyrosine-protein kinase BTK (Brutons tyrosine kinase) (Agammaglobulinaemia tyrosine kinase) (ATK) (B cell progenitor kinase) (BPK) (Kinase EMB); tyrosine-protein kinase LYK; unnamed protein product; X-linked immune deficiency

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Gene Aliases: AGMX1; AT; ATK; BPK; EMT; IGHD3; IMD1; LPFS1; LYK; PSCTK1; PSCTK2; Tcsk; Tsk; xid; XLA

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UniProt ID: (Human) Q06187, (Mouse) P35991, (Human) Q08881, (Mouse) Q03526

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Entrez Gene ID: (Human) 695, (Mouse) 12229, (Human) 3702, (Mouse) 16428

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Function(s)
protein kinase activity protein tyrosine kinase activity non-membrane spanning protein tyrosine kinase activity protein binding ATP binding phosphatidylinositol-3,4,5-trisphosphate binding phospholipase activator activity identical protein binding phospholipase binding
Process(es)
positive regulation of cytokine production NK T cell differentiation adaptive immune response protein phosphorylation signal transduction intracellular signal transduction T cell activation gamma-delta T cell activation T cell receptor signaling pathway B cell receptor signaling pathway neutrophil homeostasis positive regulation of type III hypersensitivity positive regulation of type I hypersensitivity negative regulation of cytokine production histamine secretion by mast cell positive regulation of immunoglobulin production regulation of B cell cytokine production MyD88-dependent toll-like receptor signaling pathway regulation of B cell apoptotic process mesoderm development peptidyl-tyrosine phosphorylation calcium-mediated signaling proteoglycan catabolic process positive regulation of B cell proliferation response to lipopolysaccharide negative regulation of interleukin-10 production positive regulation of interleukin-6 production positive regulation of tumor necrosis factor production cellular response to reactive oxygen species Fc-epsilon receptor signaling pathway B cell activation positive regulation of I-kappaB kinase/NF-kappaB signaling innate immune response positive regulation of B cell differentiation positive regulation of phagocytosis regulation of B cell activation negative regulation of B cell activation monocyte proliferation negative regulation of leukocyte proliferation cellular response to molecule of fungal origin apoptotic signaling pathway positive regulation of cGAS/STING signaling pathway positive regulation of interleukin-17A production positive regulation of NLRP3 inflammasome complex assembly positive regulation of synoviocyte proliferation eosinophil homeostasis B cell affinity maturation negative regulation of B cell proliferation positive regulation of type I interferon production peptidyl-tyrosine autophosphorylation protein autophosphorylation cell maturation cellular response to interleukin-7 cGAS/STING signaling pathway cellular defense response
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