Revision 1

#48697Store at -20C

1 Kit

(6 x 20 microliters)

Cell Signaling Technology

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

Support: 877-678-TECH (8324)

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For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes Product # Quantity Mol. Wt Isotype/Source
Toll-like Receptor 1 Antibody 2209 20 µl 86 kDa Rabbit 
Toll-like Receptor 2 (D7G9Z) Rabbit mAb 12276 20 µl 90-105 kDa Rabbit IgG
Toll-like Receptor 3 (D10F10) Rabbit mAb 6961 20 µl 115-130 kDa Rabbit IgG
Toll-like Receptor 6 (D1Z8B) Rabbit mAb 12717 20 µl 90-110 kDa Rabbit IgG
Toll-like Receptor 7 (D7) Rabbit mAb 5632 20 µl 140 kDa Rabbit IgG
Toll-like Receptor 8 (D3Z6J) Rabbit mAb 11886 20 µl 150 kDa Rabbit IgG
Toll-like Receptor 9 (D9M9H) XP® Rabbit mAb 13674 20 µl 130 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 Toll-like Receptor Antibody Sampler Kit II provides an economical means of detecting expression of various Toll-like receptors (TLRs). The kit contains enough primary and secondary antibodies to perform at least two western blot experiments.

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

Members of the Toll-like receptor (TLR) family, named for the closely related Toll receptor in Drosophila, play a pivotal role in innate immune responses (1-4). TLRs recognize conserved motifs found in various pathogens and mediate defense responses (5-7). TLR1, TLR2, TLR4, TLR5, TLR6, and TLR11 are localized to the plasma membrane, while TLR3, TLR7, TLR8, and TLR9 are localized to intracellular membranes including endosomal membranes. Triggering of the TLR pathway leads to the activation of NF-κB and subsequent regulation of immune and inflammatory genes (4). The TLRs and members of the IL-1 receptor family share a conserved stretch of approximately 200 amino acids known as the Toll/Interleukin-1 receptor (TIR) domain (1). Upon activation, TLRs associate with a number of cytoplasmic adaptor proteins containing TIR domains, including myeloid differentiation factor 88 (MyD88), MyD88-adaptor-like/TIR-associated protein (MAL/TIRAP), Toll-receptor-associated activator of interferon (TRIF), and Toll-receptor-associated molecule (TRAM) (8-10). This association leads to the recruitment and activation of IRAK1 and IRAK4, which form a complex with TRAF6 to activate TAK1 and IKK (8,11-14). Activation of IKK leads to the degradation of IκB, which normally maintains NF-κB in an inactive state by sequestering it in the cytoplasm. TLR1 and TLR6 associate with TLR2 to cooperatively mediate response to bacterial lipoproteins and fungal zymosan (6,15). TLR3 is an endosomal TLR that recognizes double-stranded RNA derived from viruses (7). TLR7 and TLR8 recognize single-stranded viral RNA and are also activated by synthetic imidazoquinoline compounds including R-848 (16,17). TLR9 recognizes unmethylated CpG motifs present on bacterial DNA (18).

  1. Akira, S. (2003) J Biol Chem 278, 38105-8.
  2. Beutler, B. (2004) Nature 430, 257-63.
  3. Dunne, A. and O'Neill, L.A. (2003) Sci STKE 2003, re3.
  4. Medzhitov, R. et al. (1997) Nature 388, 394-7.
  5. Schwandner, R. et al. (1999) J Biol Chem 274, 17406-9.
  6. Takeuchi, O. et al. (1999) Immunity 11, 443-51.
  7. Alexopoulou, L. et al. (2001) Nature 413, 732-8.
  8. Zhang, F.X. et al. (1999) J Biol Chem 274, 7611-4.
  9. Horng, T. et al. (2001) Nat Immunol 2, 835-41.
  10. Oshiumi, H. et al. (2003) Nat Immunol 4, 161-7.
  11. Muzio, M. et al. (1997) Science 278, 1612-5.
  12. Wesche, H. et al. (1997) Immunity 7, 837-47.
  13. Suzuki, N. et al. (2002) Nature 416, 750-6.
  14. Irie, T. et al. (2000) FEBS Lett 467, 160-4.
  15. Ozinsky, A. et al. (2000) Proc Natl Acad Sci U S A 97, 13766-71.
  16. Heil, F. et al. (2004) Science 303, 1526-9.
  17. Jurk, M. et al. (2002) Nat Immunol 3, 499.
  18. Hemmi, H. et al. (2000) Nature 408, 740-5.

Background References

    Trademarks and Patents

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

    Limited Uses

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    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 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 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.

    Revision 1
    #48697

    Toll-like Receptor Antibody Sampler Kit II

    Toll-like Receptor Antibody Sampler Kit II: Image 1 Expand Image
    Western blot analysis of extracts from THP-1 cells differentiated with TPA #4174 (80 nM, 16 hr), untreated (-) or treated with Human Interferon-γ (hIFN-γ) #8901 (20 ng/mL, 8 hr; +), using Toll-like Receptor 8 (D3Z6J) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
    Toll-like Receptor Antibody Sampler Kit II: Image 2 Expand Image
    Western blot analysis of THP-1 and MUTZ-3 cells using Toll-like Receptor 2 (D7G9Z) Rabbit mAb.
    Toll-like Receptor Antibody Sampler Kit II: Image 3 Expand Image
    Western blot analysis of extracts from THP-1 cells differentiated overnight with 80 nM TPA (12-O-Tetradecanoylphorbol-13-Acetate) #4174 and Raw 264.7 cells using Toll-like Receptor 6 (D1Z8B) Rabbit mAb.
    Toll-like Receptor Antibody Sampler Kit II: Image 4 Expand Image
    Western blot analysis of extracts from various cell lines using Toll-like Receptor 9 (D9M9H) XP® Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
    Toll-like Receptor Antibody Sampler Kit II: Image 5 Expand Image
    Western blot analysis of extracts from HeLa cells, mock transfected or transfected with human TLR1, using Toll-like Receptor 1 Antibody.
    Toll-like Receptor Antibody Sampler Kit II: Image 6 Expand Image
    Western blot analysis of extracts from various cell lines using Toll-like Receptor 7 (D7) Rabbit mAb.
    Toll-like Receptor Antibody Sampler Kit II: Image 7 Expand Image
    Western blot analysis of extracts from MCF 10A cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-) or SignalSilence® Toll-like Receptor 3 siRNA I #6236 (+), using Toll-like Receptor 3 (D10F10) Rabbit mAb #6961 (upper) or α-Tubulin (11H10) Rabbit mAb #2125 (lower). The Toll-like Receptor 3 (D10F10) Rabbit mAb confirms silencing of Toll-like Receptor 3 expression, while the α-Tubulin (11H10) Rabbit mAb is used as a loading control.
    Toll-like Receptor Antibody Sampler Kit II: 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.
    Toll-like Receptor Antibody Sampler Kit II: Image 9 Expand Image
    Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing HA-tagged full-length human TLR8 (hTLR8-HA; +), using Toll-like Receptor 8 (D3Z6J) Rabbit mAb.
    Toll-like Receptor Antibody Sampler Kit II: Image 10 Expand Image
    Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing HA-tagged full-length human TLR2 (hTLR2-HA; +), using Toll-like Receptor 2 (D7G9Z) Rabbit mAb.
    Toll-like Receptor Antibody Sampler Kit II: Image 11 Expand Image
    Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing HA-tagged full-length human TLR6 (hTLR6-HA; +) or transfected with a construct expressing HA-tagged full-length mouse TLR6 (mTLR6-HA; +), using Toll-like Receptor 6 (D1Z8B) Rabbit mAb.
    Toll-like Receptor Antibody Sampler Kit II: Image 12 Expand Image
    Immunoprecipitation of Toll-like receptor 9 from Ramos cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or Toll-like Receptor 9 (D9M9H) XP® Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using Toll-like Receptor 9 (D9M9H) XP® Rabbit mAb.
    Toll-like Receptor Antibody Sampler Kit II: Image 13 Expand Image
    Western blot analysis of extracts from SR cells using Toll-like Receptor 1 Antibody.
    Toll-like Receptor Antibody Sampler Kit II: Image 14 Expand Image
    Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with human TLR7 (+), using Toll-like Receptor 7 (D7) Rabbit mAb.
    Toll-like Receptor Antibody Sampler Kit II: Image 15 Expand Image
    Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with human TLR3 (+), using Toll-like Receptor 3 (D10F10) Rabbit mAb.
    Toll-like Receptor Antibody Sampler Kit II: Image 16 Expand Image
    Immunoprecipitation of TLR2 from MUTZ-3 cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or Toll-like Receptor 2 (D7G9Z) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using Toll-like Receptor 2 (D7G9Z) Rabbit mAb.
    Toll-like Receptor Antibody Sampler Kit II: Image 17 Expand Image
    Confocal immunofluorescent analysis of RPMI 8226 (left) and THP-1 (right) cells using Toll-like Receptor 9 (D9M9H) XP® Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    Toll-like Receptor Antibody Sampler Kit II: Image 18 Expand Image
    Western blot analysis of extracts from HT-29 cells, untreated or following transfection with pIpC (100 μg/ml; overnight), using Toll-like Receptor 3 (D10F10) Rabbit mAb.
    Toll-like Receptor Antibody Sampler Kit II: Image 19 Expand Image
    Flow cytometric analysis of THP-1 cells (blue) and RPMI 8226 cells (green) using Toll-like Receptor 9 (D9M9H) XP® Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) was used as a secondary antibody.