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72159
B Cell Signaling Antibody Sampler Kit II
Primary Antibodies
Antibody Sampler Kit

B Cell Signaling Antibody Sampler Kit II #72159

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B Cell Signaling Antibody Sampler Kit II: Image 1

Immunohistochemical analysis of paraffin-embedded human lymphoma using CD79A (D1X5C) XP® Rabbit mAb.

B Cell Signaling Antibody Sampler Kit II: Image 2

Flow cytometric analysis of human whole blood lymphocytes using CD79A (D1X5C) XP® Rabbit mAb. Cells were co-stained with CD19-APC and CD3-PE antibodies to allow for CD79A analysis in B cells (green) and T cells (blue), respectively. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

B Cell Signaling Antibody Sampler Kit II: Image 3

Flow cytometric analysis of Ramos cells, untreated (blue) or IgM-treated (green), using Phospho-CD79A (Tyr182) Antibody.

B Cell Signaling Antibody Sampler Kit II: Image 4

Immunohistochemical analysis of paraffin-embedded human endometrioid carcinoma using CD19 (D4V4B) XP® Rabbit mAb performed on the Leica® Bond™ Rx.

B Cell Signaling Antibody Sampler Kit II: Image 5

Western blot analysis of SDS extracts from untreated or anti-human IgM treated (12 µg/ml for 2 minutes) Ramos cells using Phospho-CD19 (Tyr531) Antibody (upper) and control CD19 Antibody #3574 (lower).

B Cell Signaling Antibody Sampler Kit II: Image 6

Immunoprecipitation of Phospho-Btk (Tyr223) from Ramos cells, serum-starved followed by treatment with anti-human IgM (12 μg/ml, 10 min) using Phospho-Btk (Tyr223) (D1D2Z) Rabbit mAb. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotyope Control #3900, lane 3 is Phospho-Btk (Tyr223) (D1D2Z) Rabbit mAb (1:50), and lane 4 is Phospho-Btk (Tyr223) (D1D2Z) Rabbit mAb (1:100). Western blot analysis was performed using Phospho-Btk (Tyr223) (D1D2Z) Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as the secondary antibody.

B Cell Signaling Antibody Sampler Kit II: Image 7

Flow cytometric analysis of RL-7 cells using Syk (D3Z1E) XP® Rabbit mAb (blue) compared to Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (red). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) #4414 was used as a secondary antibody.

B Cell Signaling Antibody Sampler Kit II: Image 8

Flow cytometric analysis of Ramos cells, untreated (blue) or treated with anti-IgM (green), using Phospho-Syk(Tyr525/526) (C87C1) Rabbit mAb, or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (PE Conjugate) #8885 was used as a secondary antibody.

B Cell Signaling Antibody Sampler Kit II: Image 9

Western blot analysis of SDS extracts from control or anti-human IgM treated (12 µg/ml for 2 minutes) Ramos cells using Phospho-BLNK (Tyr96) Antibody.

B Cell Signaling Antibody Sampler Kit II: Image 10

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.

B Cell Signaling Antibody Sampler Kit II: Image 11

Immunohistochemical analysis of paraffin-embedded human squamous cell lung carcinoma using Btk (D3H5) Rabbit mAb performed on the Leica® Bond™ Rx.

B Cell Signaling Antibody Sampler Kit II: Image 12

After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.

B Cell Signaling Antibody Sampler Kit II: Image 13

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using CD79A (D1X5C) XP® Rabbit mAb.

B Cell Signaling Antibody Sampler Kit II: Image 14

Confocal immunofluorescent analysis of Ramos cells, untreated (left) or IgM-treated (middle), and Jurkat cells (right) using Phospho-CD79A (Tyr182) Antibody (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

B Cell Signaling Antibody Sampler Kit II: Image 15

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using CD19 (D4V4B) XP® Rabbit mAb.

B Cell Signaling Antibody Sampler Kit II: Image 16

Western blot analysis of extracts from Daudi cells, serum-starved overnight, then vehicle-treated (lane 1), treated with anti-human IgM (12 μg/ml, 10 min; lane 2), or pre-treated with Ibrutinib #16483 (1 μM, 60 min) prior to anti-IgM treatment (lane 3), using Phospho-Btk (Tyr23) (D1D2Z) Rabbit mAb (upper) or Btk (D6H5) Rabbit mAb #8547 (lower).

B Cell Signaling Antibody Sampler Kit II: Image 17

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Syk (D3Z1E) XP® Rabbit mAb.

B Cell Signaling Antibody Sampler Kit II: Image 18

Confocal immunofluorescent analysis of Ramos cells, serum-starved (overnight; left) or IgM-treated (12 ug/ml, 2 minutes; right), using Phospho-Syk (Tyr525/526) (C87C1) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

B Cell Signaling Antibody Sampler Kit II: Image 19

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.

B Cell Signaling Antibody Sampler Kit II: Image 20

Western blot analysis of extracts from Ramos cells untreated or treated with anti-human IgM (12 µg/ml for 10 minutes), using Phospho-CD79A (Tyr182) Antibody (upper) or CD79A Antibody #3351 (lower).

B Cell Signaling Antibody Sampler Kit II: Image 21

Immunohistochemical analysis of paraffin-embedded human lung carcinoma using CD19 (D4V4B) XP® Rabbit mAb.

B Cell Signaling Antibody Sampler Kit II: Image 22

Immunohistochemical analysis of paraffin-embedded human lymph node using Syk (D3Z1E) XP® Rabbit mAb.

B Cell Signaling Antibody Sampler Kit II: Image 23

Western blot analysis of extracts from Ramos cells, untreated or treated with anti-IgM, using Phospho-Syk (Tyr525/526) (C87C1) Rabbit mAb (upper) or Syk Antibody #2712 (lower).

B Cell Signaling Antibody Sampler Kit II: Image 24

Immunohistochemical analysis of paraffin-embedded human lung carcinoma using CD79A (D1X5C) XP® Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).

B Cell Signaling Antibody Sampler Kit II: Image 25

Immunohistochemical analysis of paraffin-embedded human lymphoma using CD19 (D4V4B) XP® Rabbit mAb.

B Cell Signaling Antibody Sampler Kit II: Image 26

Immunohistochemical analysis of paraffin-embedded mouse spleen using Syk (D3Z1E) XP® Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).

B Cell Signaling Antibody Sampler Kit II: Image 27

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Btk (D3H5) Rabbit mAb. Note staining of inflammatory cells.

B Cell Signaling Antibody Sampler Kit II: Image 28

Western blot analysis of extracts from various cell lines using CD79A (D1X5C) XP® Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).

B Cell Signaling Antibody Sampler Kit II: Image 29

Immunohistochemical analysis of paraffin-embedded mouse spleen using CD19 (D4V4B) XP® Rabbit mAb.

B Cell Signaling Antibody Sampler Kit II: Image 30

Immunoprecipitation of Syk protein from SR cell extracts, using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or Syk(D3Z1E) XP® Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using Syk (D3Z1E) XP® Rabbit mAb

B Cell Signaling Antibody Sampler Kit II: Image 31

Immunohistochemical analysis of paraffin-embedded human B-cell lymphoma using Btk (D3H5) Rabbit mAb.

B Cell Signaling Antibody Sampler Kit II: Image 32

Western blot analysis of extracts from Ramos cells, untreated (-) or PNGase F-treated (+), using CD79A (D1X5C) XP® Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).

B Cell Signaling Antibody Sampler Kit II: Image 33

Immunohistochemical analysis of paraffin-embedded human tonsil using CD19 (D4V4B) XP® Rabbit mAb.

B Cell Signaling Antibody Sampler Kit II: Image 34

Western blot analysis of extracts from various cell lines using Syk (D3Z1E) XP® Rabbit mAb.

B Cell Signaling Antibody Sampler Kit II: Image 35

Immunohistochemical analysis of paraffin-embedded mouse colon using Btk (D3H5) Rabbit mAb.

B Cell Signaling Antibody Sampler Kit II: Image 36

Western blot analysis of extracts from various cell lines using CD19 (D4V4B) XP® Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower). As expected, CD19 protein is not detected in either U-937 cells or Raw 264.7 cells.

B Cell Signaling Antibody Sampler Kit II: Image 37

Immunohistochemical analysis of paraffin-embedded human ovarian carcinoma using Btk (D3H5) Rabbit mAb. Note staining of inflammatory cells.

B Cell Signaling Antibody Sampler Kit II: Image 38

Western blot analysis of extracts from Daudi cells, untreated (-) or treated with PNGase F (+), using CD19 (D4V4B) XP® Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).

B Cell Signaling Antibody Sampler Kit II: Image 39

Immunohistochemical analysis of paraffin-embedded cell pellets, Ramos(left) or Jurkat (right), using Btk (D3H5) Rabbit mAb.

B Cell Signaling Antibody Sampler Kit II: Image 40

Western blot analysis of extracts from various cell lines using Btk (D3H5) Rabbit mAb.

To Purchase # 72159T
Product # Size Price
72159T
1 Kit  (9 x 20 µl) N/A

Product Description

The B Cell Signaling Antibody Sampler Kit II provides an economical means to examine key signaling proteins commonly associated with B cell activation. The provided antibodies allow monitoring of both total protein levels and the phosphorylation state. The kit includes enough antibody to perform two western blot experiments with each primary antibody.

Specificity / Sensitivity

All antibodies contained in this kit detect endogenous levels of their respective target protein.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic peptide and are purified by protein A and peptide affinity chromatography. Monoclonal antibodies are produced by immunizing animals with recombinant human proteins or synthetic peptides.

Background

Antigen receptors found on the surface of B cells contain a heterodimeric signaling component composed of CD79A and CD79B, also known as Ig α and Ig ß, respectively. Presence of this receptor complex is essential for B-cell development and function. Antigen binding precedes formation of the CD79A and CD79B heterodimer and subsequent activation of receptor associated kinases. Tyr182 of mouse CD79A (corresponding to Tyr188 of human CD79A) is one of two key tyrosine residues in the immunoreceptor tyrosine-based activation motif (ITAM) of CD79A that are phosphorylated by Src family kinases (e.g., Lyn, Blk), and play a critical role in modulating signal transduction following immune receptor activation.

Syk is a protein tyrosine kinase that plays an important role in intracellular signal transduction in hematopoietic cells (1-3). Syk interacts with immunoreceptor tyrosine-based activation motifs (ITAMs) located in the cytoplasmic domains of immune receptors (4). It couples the activated immunoreceptors to downstream signaling events that mediate diverse cellular responses, including proliferation, differentiation, and phagocytosis (4). There is also evidence that Syk plays a role in nonimmune cells; Syk is a potential tumor suppressor in human breast carcinomas (5). Tyrosine 525 and 526 are located in the activation loop of the Syk kinase domain, and phosphorylation of Tyr525/526 of human Syk (equivalent to the Tyr519/520 of mouse Syk) is essential for Syk function (6).

Lyn, one of the Src family members, is predominantly expressed in hematopoietic cells (7). Two tyrosine residues have been reported to play a crucial role in the regulation of protein tyrosine kinases of the Src family. Autophosphorylation of Tyr396 (equivalent to Tyr416 of Src), located in the catalytic domain, correlates with enzyme activation. Csk-mediated phosphorylation of the carboxy-terminal Tyr507 (equivalent to Tyr527 of Src) inactivates the kinase. Tyrosine phosphorylation and activation of Lyn occurs upon association with cell surface receptors such as the B cell Ag receptor (BCR) and CD40 (8-10).

Bruton's tyrosine kinase (Btk) is a member of the Btk/Tec family of cytoplasmic tyrosine kinases. Btk plays an important role in B cell development (11,12). 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 (13-15). The membrane-located 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 kinase, leading to autophosphorylation at Tyr223 within the SH3 domain, which is necessary for full activation (16,17).

CD19 is a 95 kDa coreceptor that amplifies the signaling cascade in B cells (18). On the B cell surface, CD19 associates with CD21, CD81, and Leu-13 to exert its function. The cytoplasmic tail of CD19 has nine conserved tyrosine residues playing critical roles in CD19-mediated function by coupling signaling molecules to the receptor (18). After BCR or CD19 ligation, Tyr531 and Tyr500 of CD19 are progressively phosphorylated. This phosphorylation enables the coupling of PI3 kinase and Src family tyrosine kinase to CD19 and activates the PI3K and Src signaling pathways (19,20).

B cell linker protein (BLNK), also known as SLP-65 or BASH, is an adaptor molecule that plays key roles in B cell activation and B cell antigen receptor (BCR) engagement. BLNK acts at the interface between BCR-associated Syk and downstream signaling cascades.

  1. Cheng, A.M. and Chan, A.C. (1997) Curr Opin Immunol 9, 528-33.
  2. Chu, D.H. et al. (1998) Immunol Rev 165, 167-80.
  3. Yamanashi, Y. et al. (1989) Proc Natl Acad Sci U S A 86, 6538-42.
  4. Khan, W.N. (2001) Immunol Res 23, 147-56.
  5. Tedder, T.F. et al. (1997) Immunity 6, 107-18.
  6. Kurosaki, T. (1997) Curr Opin Immunol 9, 309-18.
  7. Yamanashi, Y. et al. (1991) Science 251, 192-4.
  8. Lewis, C.M. et al. (2001) Curr Opin Immunol 13, 317-25.
  9. Buhl, A.M. and Cambier, J.C. (1999) J Immunol 162, 4438-46.
  10. Burkhardt, A.L. et al. (1991) Proc Natl Acad Sci U S A 88, 7410-4.
  11. Salim, K. et al. (1996) EMBO J 15, 6241-50.
  12. Fujimoto, M. et al. (2000) Immunity 13, 47-57.
  13. Turner, M. et al. (2000) Immunol Today 21, 148-54.
  14. Ren, C.L. et al. (1994) J Exp Med 179, 673-80.
  15. Rameh, L.E. et al. (1997) J Biol Chem 272, 22059-66.
  16. Coopman, P.J. et al. (2000) Nature 406, 742-7.
  17. Várnai, P. et al. (1999) J Biol Chem 274, 10983-9.
  18. Rawlings, D.J. et al. (1996) Science 271, 822-5.
  19. Park, H. et al. (1996) Immunity 4, 515-25.
  20. Zhang, J. et al. (2000) J Biol Chem 275, 35442-7.

Pathways & Proteins

Explore pathways + proteins related to this product.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST's products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST's Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.
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