Revision 1

#8347Store at -20C

1 Kit

(4 x 20 microliters)

Cell Signaling Technology

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

Support: 877-678-TECH (8324)

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3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes Product # Quantity Mol. Wt Isotype/Source
TRAF1 (45D3) Rabbit mAb 4715 20 µl 50 kDa Rabbit 
TRAF2 (C192) Antibody 4724 20 µl 53 kDa Rabbit 
TRAF3 Antibody 4729 20 µl 62 kDa Rabbit 
TRAF6 (D21G3) Rabbit mAb 8028 20 µl 60 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 TRAF Antibody Sampler Kit provides an economical means to evaluate endogenous levels of TRAF1, 2, 3, and 6. The kit contains enough primary and secondary antibodies to perform two western mini-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

TRAFs (TNF receptor-associated factors) are a family of multifunctional adaptor proteins that bind to surface receptors and recruit additional proteins to form multiprotein signaling complexes capable of promoting cellular responses (1-3). Members of the TRAF family share a common carboxy-terminal "TRAF domain", which mediates interactions with associated proteins; many also contain amino-terminal Zinc/RING finger motifs. The first TRAFs identified, TRAF1 and TRAF2, were found by virtue of their interactions with the cytoplasmic domain of TNF-receptor 2 (TNFRII) (4). The six known TRAFs (TRAF1-6) act as adaptor proteins for a wide range of cell surface receptors and participate in the regulation of cell survival, proliferation, differentiation, and stress responses.
While TRAF2 was originally described through its interaction with TNFRII, it has since been shown to interact with other surface receptors including CD27, CD30, CD40, 4-1BB, Ox40, HVEM/ATAR, and LMP-1 (1-3). TRAF2 also associates with a large number of intracellular proteins, including TRADD, FADD, I-TRAF/TANK, TRIP, A20, c-IAP1 and 2, Casper, RIP, and NIK, which help to regulate cell survival. Dominant negative and knockout studies have shown that TRAF2 plays an important role in TNF-mediated activation of NF-κB and the MAPK/JNK kinase pathway (5-7).

TRAF6 plays a critical role in innate and adaptive immunity, bone metabolism, and development of certain tissues including the nervous system (8). TRAF6 deficiency results in osteopetrosis and defective IL-1, CD40, and LPS signaling (9) as well as defects in neuronal development (10). Unlike other TRAF family members that mediate signaling through TNF, TRAF6 has unique binding activities (11) that result in signaling responses from the interleukin-1 receptor (IL-1R) (12), toll-like receptor (13,14), CD (15), RANK (16,17), and p75 neurotrophin receptor (18). TRAF6 associates directly with CD40 and RANK, and indirectly with IL-1R/TLR through IRAK (13). This leads to activation of NF-κB and MAP kinase signaling pathways through downstream association with the TAB/TAK-1 complex (19). TRAF6 also activates Src family nonreceptor tyrosine kinases leading to Akt activation (20).

  1. Arch, R.H. et al. (1998) Genes Dev 12, 2821-30.
  2. Chung, J.Y. et al. (2002) J Cell Sci 115, 679-88.
  3. Bradley, J.R. and Pober, J.S. (2001) Oncogene 20, 6482-91.
  4. Rothe, M. et al. (1994) Cell 78, 681-92.
  5. Yeh, W.C. et al. (1997) Immunity 7, 715-25.
  6. Reinhard, C. et al. (1997) EMBO J 16, 1080-92.
  7. Rothe, M. et al. (1995) Science 269, 1424-7.
  8. Wu, H. and Arron, J.R. (2003) Bioessays 25, 1096-105.
  9. Lomaga, M.A. et al. (1999) Genes Dev 13, 1015-24.
  10. Lomaga, M.A. et al. (2000) J Neurosci 20, 7384-93.
  11. Ye, H. et al. (2002) Nature 418, 443-7.
  12. Cao, Z. et al. (1996) Nature 383, 443-6.
  13. Muzio, M. et al. (1997) Science 278, 1612-5.
  14. Medzhitov, R. et al. (1998) Mol Cell 2, 253-8.
  15. Ishida, T. et al. (1996) J Biol Chem 271, 28745-8.
  16. Darnay, B.G. et al. (1998) J Biol Chem 273, 20551-5.
  17. Wong, B.R. et al. (1998) J Biol Chem 273, 28355-9.
  18. Khursigara, G. et al. (1999) J Biol Chem 274, 2597-600.
  19. Ninomiya-Tsuji, J. et al. (1999) Nature 398, 252-6.
  20. Wong, B.R. et al. (1999) Mol Cell 4, 1041-9.

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.

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    Revision 1
    #8347

    TRAF Antibody Sampler Kit

    TRAF Antibody Sampler Kit: Image 1 Expand Image
    Simple Western™ analysis of lysates (0.1 mg/mL) from 293T cells using TRAF2 (C192) Antibody #4724. 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.
    TRAF Antibody Sampler Kit: Image 2 Expand Image
    Western blot analysis of extracts from Raji and SR cells, using TRAF1 (45D3) Rabbit mAb.
    TRAF Antibody Sampler Kit: Image 3 Expand Image
    Western blot analysis of extracts from Jurkat, 293, HeLa, C2C12, and NIH/3T3 cells, using TRAF2 (C192) Antibody.
    TRAF Antibody Sampler Kit: Image 4 Expand Image
    Western blot analysis of extracts from COS-7 cells, untransfected (-) or transfected with human TRAF3 construct (+) using TRAF3 Antibody.
    TRAF Antibody Sampler Kit: Image 5 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.
    TRAF Antibody Sampler Kit: Image 6 Expand Image
    Western blot analysis of extracts from 293T cells, either mock transfected (-) or transfected with a cDNA expression construct encoding full-length human TRAF6 (+), using TRAF6 (D21G3) Rabbit mAb.
    TRAF Antibody Sampler Kit: Image 7 Expand Image
    Immunohistochemical analysis of paraffin-embedded human tonsil using TRAF1 (45D3) Rabbit mAb preincubated with a control peptide (left) or TRAF1 Blocking Peptide #1066 (right).
    TRAF Antibody Sampler Kit: Image 8 Expand Image
    Western blot analysis of extracts from various cell lines using TRAF3 Antibody.
    TRAF Antibody Sampler Kit: Image 9 Expand Image
    Western blot analysis of extracts from various cell lines using TRAF6 (D21G3) Rabbit mAb.
    TRAF Antibody Sampler Kit: Image 10 Expand Image
    Confocal immunofluorescent analysis of HT-1080 (upper) and HEK/293 (lower) cells, untreated (left) or treated with TNF-α #8902 (right), using TRAF1 (45D3) Rabbit mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    TRAF Antibody Sampler Kit: Image 11 Expand Image
    Immunoprecipitation of TRAF3 from HDLM-2 cell extracts. Lane 1 is Normal Rabbit IgG #2729, lane 2 is 10% input, and lane 3 is TRAF3 Antibody #4729. Western blot analysis was performed using TRAF3 Antibody #4729. Mouse Anti-rabbit (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 was used as the secondary antibody.
    TRAF Antibody Sampler Kit: Image 12 Expand Image
    Flow cytometric analysis of Raji cells, using TRAF1 (45D3) Rabbit mAb (blue) compared to a nonspecific negative control antibody (red).