Revision 1

#9382Store 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.
Product Includes Product # Quantity Mol. Wt Isotype/Source
Zap-70 (D1C10E) XP® Rabbit mAb 3165 40 µl 70 kDa Rabbit IgG
Phospho-Zap-70 (Tyr319)/Syk (Tyr352) (65E4) Rabbit mAb 2717 40 µl 70, 72 kDa Rabbit IgG
SLP-76 Antibody 4958 40 µl 76 kDa Rabbit 
Phospho-LAT (Tyr220) Antibody 3584 40 µl 36, 38 kDa Rabbit 
LAT Antibody 9166 40 µl 36, 38 kDa Rabbit 
Phospho-Lck (Tyr505) Antibody 2751 40 µl 56 kDa Rabbit 
Lck (73A5) Rabbit mAb 2787 40 µl 56 kDa Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 

Please visit cellsignal.com for individual component applications, species cross-reactivity, dilutions, protocols, and additional product information.

Description

The T Cell Signaling Antibody Sampler Kit contains primary and secondary antibodies to perform four Western blots with each antibody.

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.

Background

The Syk family protein tyrosine kinase Zap-70 is expressed in T and NK cells and plays a critical role in mediating T cell activation in response to T cell receptor (TCR) engagement (1). Following TCR engagement, Zap-70 is rapidly phosphorylated on several tyrosine residues through autophosphorylation and transphosphorylation by the Src family tyrosine kinase Lck (2-6). Tyrosine phosphorylation correlates with increased Zap-70 kinase activity and downstream signaling events. Expression of Zap-70 is correlated with disease progression and survival in patients with chronic lymphocytic leukemia (7,8).
LAT, a transmembrane adaptor protein expressed in T, NK and mast cells, is an important mediator for T cell receptor (TCR) signaling (9). Upon TCR engagement, activated Zap-70 phosphorylates LAT at multiple conserved tyrosine residues within SH2 binding motifs, exposing these motifs as the docking sites for downstream signaling targets (10,11). The phosphorylation of LAT at Tyr171 and 191 enables the binding of Grb2, Gads/SLP-76, PLCgamma1 and PI3 kinase through their SH2 domain and translocates them to the membrane. This process eventually leads to activation of the corresponding signaling pathways (11-12).

  1. Chu, D.H. et al. (1998) Immunol Rev 165, 167-80.
  2. Iwashima, M. et al. (1994) Science 263, 1136-9.
  3. Neumeister, E.N. et al. (1995) Mol Cell Biol 15, 3171-8.
  4. Chan, A.C. et al. (1995) EMBO J 14, 2499-508.
  5. Williams, B.L. et al. (1999) EMBO J 18, 1832-44.
  6. Di Bartolo, V. et al. (1999) J Biol Chem 274, 6285-94.
  7. Wiestner, A. et al. (2003) Blood 101, 4944-51.
  8. Crespo, M. et al. (2003) N Engl J Med 348, 1764-75.
  9. Wonerow, P. and Watson, S.P. (2001) Oncogene 20, 6273-83.
  10. Zhang, W. et al. (1998) Cell 92, 83-92.
  11. Paz, P.E. et al. (2001) Biochem J 356, 461-71.
  12. Zhang, W. et al. (2000) J Biol Chem 275, 23355-61.

Background References

    Trademarks and Patents

    Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
    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

    法的な権限を与えられたCSTの担当者が署名した書面によって別途明示的に合意された場合を除き、 CST、その関連会社または代理店が提供する製品には以下の条件が適用されます。お客様が定める条件でここに定められた条件に含まれるものを超えるもの、 または、ここに定められた条件と異なるものは、法的な権限を与えられたCSTの担当者が別途書面にて受諾した場合を除き、拒絶され、 いかなる効力も効果も有しません。

    研究専用 (For Research Use Only) またはこれに類似する表示がされた製品は、 いかなる目的についても FDA または外国もしくは国内のその他の規制機関により承認、認可または許可を受けていません。 お客様は製品を診断もしくは治療目的で使用してはならず、また、製品に表示された内容に違反する方法で使用してはなりません。 CST が販売または使用許諾する製品は、エンドユーザーであるお客様に対し、使途を研究および開発のみに限定して提供されるものです。 診断、予防もしくは治療目的で製品を使用することまたは製品を再販売 (単独であるか他の製品等の一部であるかを問いません) もしくはその他の商業的利用の目的で購入することについては、CST から別途許諾を得る必要があります。 お客様は以下の事項を遵守しなければなりません。(a) CST の製品 (単独であるか他の資材と一緒であるかを問いません) を販売、使用許諾、貸与、寄付もしくはその他の態様で第三者に譲渡したり使用させたりしてはなりません。また、商用の製品を製造するために CST の製品を使用してはなりません。(b) 複製、改変、リバースエンジニアリング、逆コンパイル、 分解または他の方法により製品の構造または技術を解明しようとしてはなりません。また、 CST の製品またはサービスと競合する製品またはサービスを開発する目的で CST の製品を使用してはなりません。(c) CST の製品の商標、商号、ロゴ、特許または著作権に関する通知または表示を除去したり改変したりしてはなりません。(d) CST の製品をCST 製品販売条件(CST’s Product Terms of Sale) および該当する書面のみに従って使用しなければなりません。(e) CST の製品に関連してお客様が使用する第三者の製品またはサービスに関する使用許諾条件、 サービス提供条件またはこれに類する合意事項を遵守しなければなりません。

    Revision 1
    #9382

    T Cell Signaling Antibody Sampler Kit

    T Cell Signaling Antibody Sampler Kit: Image 1 Expand Image
    Simple Western™ analysis of lysates (0.01 mg/mL) from Jurkat cells using Zap-70 (D1C10E) XP® Rabbit mAb #3165. The virtual lane view (left) shows the 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.
    T Cell Signaling Antibody Sampler Kit: Image 2 Expand Image
    Western blot analysis of extracts from Jurkat cells treated with hydrogen peroxide (2mM for 2 minutes) or with lambda phosphatase and extracts from Ramos cells treated with anti-human IgM (12 micrograms/ml for 2 minutes) using Phospho-Zap-70 (Tyr319)/Syk (Tyr352) (65E4) Rabbit mAb.
    T Cell Signaling Antibody Sampler Kit: Image 3 Expand Image
    Western blot analysis of extracts from Jurkat cells (starved for 16 hours) treated with calf intestinal alkaline phosphatase (CIP) or H2O2 (2 mM), using Phospho-Lck (Tyr505) Antibody (upper) or control Lck Antibody #2752 (lower).
    T Cell Signaling Antibody Sampler Kit: Image 4 Expand Image
    Western blot analysis of Jurkat cell extracts using Lck (73A5) Rabbit mAb.
    T Cell Signaling Antibody Sampler Kit: Image 5 Expand Image
    Western blot analysis of extracts from Jurkat cells using Zap-70 (D1C10E) XP® Rabbit mAb.
    T Cell Signaling Antibody Sampler Kit: Image 6 Expand Image
    Western blot analysis of SDS extracts from untreated or anti-CD3 treated (10 µg/ml for 2 minutes) human Jurkat cells after overnight serum starvation using Phospho-LAT (Tyr220) Antibody.
    T Cell Signaling Antibody Sampler Kit: Image 7 Expand Image
    Western blot analysis of extracts from Jurkat and BW5147cells using SLP-76 Antibody.
    T Cell Signaling Antibody Sampler Kit: Image 8 Expand Image
    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.
    T Cell Signaling Antibody Sampler Kit: Image 9 Expand Image
    Western blot analysis of extracts from Jurkat cells, untreated or treated with lambda phosphatase, using LAT Antibody.
    T Cell Signaling Antibody Sampler Kit: Image 10 Expand Image
    Confocal immunofluorescent analysis of Ramos cells, human IgM treated (left) or untreated (right), using Phospho-Zap-70 (Tyr319)/Syk (Tyr352) (65E4) Rabbit mAb (green). Red = Propidium Iodide (PI)/RNase Staining Solution #4087.
    T Cell Signaling Antibody Sampler Kit: Image 11 Expand Image
    Confocal immunofluorescent analysis of a Jurkat cell nucleus using Lck (73A5) Rabbit mAb (red) and CD3 antibody (green). Blue=DRAQ5® #4084 fluorescent DNA dye.
    T Cell Signaling Antibody Sampler Kit: Image 12 Expand Image
    Immunoprecipitation of Zap-70 from Jurkat cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Zap-70 (D1C10E) XP® Rabbit mAb. Western blot analysis was performed using Zap-70 (D1C10E) XP® Rabbit mAb. Mouse Anti-rabbit (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 was used as the secondary antibody.
    T Cell Signaling Antibody Sampler Kit: Image 13 Expand Image
    Immunohistochemical analysis of paraffin-embedded Jurkat cells, using LAT Antibody in the presence of control peptide (left) or antigen-specific peptide (right).
    T Cell Signaling Antibody Sampler Kit: Image 14 Expand Image
    Flow cytometric analysis of Ramos cells, untreated (blue) or treated with anti-IgM (12 µg/mL, 2 min; green), using Phospho-Zap-70 (Tyr319)/Syk (Tyr352) (65E4) Rabbit mAb (solid lines) or 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.
    T Cell Signaling Antibody Sampler Kit: Image 15 Expand Image
    Flow cytometric analysis of Jurkat cells, using Lck (73A5) Rabbit mAb (blue) compared to a nonspecific negative control antibody (red).
    T Cell Signaling Antibody Sampler Kit: Image 16 Expand Image
    Immunohistochemical analysis of paraffin-embedded human tonsil, showing membrane localization, using LAT Antibody.
    T Cell Signaling Antibody Sampler Kit: Image 17 Expand Image
    Confocal immunofluorescent analysis of Jurkat cells (left) and Ramos cells (right) using Zap-70 (D1C10E) XP® Rabbit mAb #3165 (green). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
    T Cell Signaling Antibody Sampler Kit: Image 18 Expand Image
    Flow cytometric analysis of Jurkat cells, using LAT Antibody (blue) compared to a nonspecific negative control antibody (red).
    T Cell Signaling Antibody Sampler Kit: Image 19 Expand Image
    Flow cytometric analysis of human peripheral blood mononuclear cells using Zap-70 (D1C10E) XP® Rabbit mAb (right) and co-stained with CD3 (UCHT1) Mouse mAb (APC Conjugate) #19881, compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (left). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.