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Phospho-BTK/ITK (Tyr551, Tyr511) Monoclonal Antibody (M4G3LN), PerCP-eFluor™ 710, eBioscience™
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FIGURE: 1 / 1
Phospho-BTK/ITK (Tyr551, Tyr511) Antibody (46-9015-41) in Flow
Intracellular staining of unstimulated (orange histogram), 5-minute F (ab')2 Anti-Mouse IgM, u chain specific Functional Grade Purified-stimulated (Product # 16-5092-85) (purple histogram), or hydorgen peroxide-activated sodium pervanadate-treated (green histogram) CD3-gated (left) or B220-gated (right) mouse splenocytes with Anti-Human/Mouse phospho-BTK/ITK (Y551/Y511) PerCP-eFluor® 710.
Product Details
46-9015-41
Product Specifications
Host/Isotype
Mouse
/ IgG2b, kappa
Recommended Isotype Control
Class
Monoclonal
Type
Antibody
Clone
M4G3LN
Conjugate
Excitation/Emission Max
482/708 nm
View spectra
Form
Liquid
Concentration
5 µL/Test
Storage conditions
4°C, store in dark, DO NOT FREEZE!
Shipping conditions
Wet ice
RRID
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.
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