Revision 8

#8547Store at -20C

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:

WB, W-S, IP, IHC-Bond, IHC-P, FC-FP

REACTIVITY:

H M

SENSITIVITY:

Endogenous

MW (kDa):

77

Source/Isotype:

Rabbit IgG

UniProt ID:

#Q06187

Entrez-Gene Id:

695

Product Information

Product Usage Information

Application Dilution
Western Blotting 1:1000
Simple Western™ 1:10 - 1:50
Immunoprecipitation 1:200
IHC Leica Bond 1:50 - 1:200
Immunohistochemistry (Paraffin) 1:50 - 1:200
Flow Cytometry (Fixed/Permeabilized) 1:100 - 1:400

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

For a carrier free (BSA and azide free) version of this product see product #94988.

Specificity / Sensitivity

Btk (D3H5) Rabbit mAb recognizes endogenous levels of total Btk protein.

Species Reactivity:

Human, Mouse

Species predicted to react based on 100% sequence homology

Rat, Hamster, Bovine, Dog, Pig, Horse

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Asp195 of human Btk protein.

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).

  1. Khan, W.N. (2001) Immunol Res 23, 147-56.
  2. Lewis, C.M. et al. (2001) Curr Opin Immunol 13, 317-25.
  3. Salim, K. et al. (1996) EMBO J 15, 6241-50.
  4. Rameh, L.E. et al. (1997) J Biol Chem 272, 22059-66.
  5. Várnai, P. et al. (1999) J Biol Chem 274, 10983-9.
  6. Rawlings, D.J. et al. (1996) Science 271, 822-5.
  7. Park, H. et al. (1996) Immunity 4, 515-25.
  8. Kang, S.W. et al. (2001) EMBO J 20, 5692-702.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Western Blot Buffer

IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

WB: Western Blotting W-S: Simple Western™ IP: Immunoprecipitation IHC-Bond: IHC Leica Bond IHC-P: Immunohistochemistry (Paraffin) FC-FP: Flow Cytometry (Fixed/Permeabilized)

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Alexa Fluor is a registered trademark of Life Technologies Corporation.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

使用に関する制限

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Revision 8
#8547

Btk (D3H5) Rabbit mAb

Western Blotting Image 1: Btk (D3H5) Rabbit mAb Expand Image
Western blot analysis of extracts from various cell lines using Btk (D3H5) Rabbit mAb.
Western Blotting Image 1: Btk (D3H5) Rabbit mAb Expand Image
Simple Western™ analysis of lysates (0.1 mg/mL) from Raji cells using Btk (D3H5) Rabbit mAb #8547. The virtual lane view (left) shows a single target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
No image available
Immunohistochemistry Image 1: Btk (D3H5) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human squamous cell lung carcinoma using Btk (D3H5) Rabbit mAb performed on the Leica® Bond Rx.
Immunohistochemistry Image 1: Btk (D3H5) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Btk (D3H5) Rabbit mAb. Note staining of inflammatory cells.
Immunohistochemistry Image 2: Btk (D3H5) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human B-cell lymphoma using Btk (D3H5) Rabbit mAb.
Immunohistochemistry Image 3: Btk (D3H5) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded mouse colon using Btk (D3H5) Rabbit mAb.
Immunohistochemistry Image 4: Btk (D3H5) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human ovarian carcinoma using Btk (D3H5) Rabbit mAb. Note staining of inflammatory cells.
Immunohistochemistry Image 5: Btk (D3H5) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded cell pellets, Ramos(left) or Jurkat (right), using Btk (D3H5) Rabbit mAb.
Flow Cytometry Image 1: Btk (D3H5) Rabbit mAb Expand Image
Human whole blood was fixed, lysed, and permeabilized as per the Cell Signaling Technology Flow Alternate Protocol and stained using Btk (D3H5) Rabbit mAb. Samples were co-stained using CD3-PE and CD19-APC to distinguish T and B cell subpopulations, respectively. B (red) and T (blue) cell population gates were applied to a histogram depicting the mean fluorescence intensity of Btk. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Flow Cytometry Image 2: Btk (D3H5) Rabbit mAb Expand Image
Flow cytometric analysis of Daudi cells (green) and Jurkat cells (blue) using Btk (D3H5) Rabbit mAb (solid lines) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab’)2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.