Cell Signaling Technology
XP Monoclonal Antibody

Product Pathways - Growth Factors/Cytokines

TNF-α (D2D4) XP® Rabbit mAb (Rodent Specific) #11948

Applications Reactivity Sensitivity MW (kDa) Isotype
W IP IF-IC F M R Endogenous 17,25,28 Rabbit

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry
Reactivity Key:  M=Mouse  R=Rat
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

* Product-specific protocol.

Specificity / Sensitivity

TNF-α (D2D4) XP® Rabbit mAb (Rodent Specific) recognizes endogenous levels of total TNF-α protein.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a recombinant mouse TNF-α protein.

Western Blotting

Western Blotting

Western blot analysis of extracts from Raw 264.7 cells, untreated (-) or treated (+) with LPS (100 ng/mL, 6 hr) and Brefeldin A #9972 (300 ng/mL, last 3 hr of stimulation), using TNF-α (D2D4) XP® Rabbit mAb (Rodent Specific) (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

Western Blotting

Western Blotting

Western blot analysis of 1 ng recombinant mouse TNF-α #5178 and 1 ng recombinant rat TNF-α using TNF-α (D2D4) XP® Rabbit mAb (Rodent Specific).

IP

IP

Immunoprecipitation of TNF-α from Raw 264.7 cell extracts, treated with LPS (100 ng/mL, 6 hr) and Brefeldin A #9972 (300 ng/mL, last 3 hr of stimulation), using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or TNF-α (D2D4) XP® Rabbit mAb (Rodent Specific) (lane 3). Lane 1 is 10% input. Western blot analysis was performed using TNF-α (D2D4) XP® Rabbit mAb (Rodent Specific). Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) #3678 and Anti-mouse IgG, HRP-linked Antibody #7076 were used as sencondary antibodies.


Flow Cytometry

Flow Cytometry

Flow cytometric analysis of Raw 264.7 cells, untreated (blue) or treated with LPS (100 ng/ml, 6 hr) and Brefeldin A #9972 (300 ng/ml, last 3 hr of treatment) (green), using TNF-α (D2D4) XP® Rabbit mAb (Rodent Specific). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) #4414 was used as a secondary antibody.

IF-IC

IF-IC

Confocal immunofluorescent analysis of Raw 264.7 cells, treated with LPS (100 ng/mL, 6 hr; left) or untreated (right), using TNF-α (D2D4) XP® Rabbit mAb (Rodent Specific) (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Background

TNF-α, the prototypical member of the TNF protein superfamily, is a homotrimeric type-II membrane protein (1,2). Membrane-bound TNF-α is cleaved by the metalloprotease TACE/ADAM17 to generate a soluble homotrimer (2). Both membrane and soluble forms of TNF-α are biologically active. TNF-α is produced by a variety of immune cells including T cells, B cells, NK cells, and macrophages (1). Cellular response to TNF-α is mediated through interaction with receptors TNF-R1 and TNF-R2 and results in activation of pathways that favor both cell survival and apoptosis depending on the cell type and biological context. Activation of kinase pathways (including JNK, Erk1/2, p38 MAPK, and NF-κB) promotes the survival of cells, while TNF-α-mediated activation of caspase-8 leads to programmed cell death (1,2). TNF-α plays a key regulatory role in inflammation and host defense against bacterial infection, notably Mycobacterium tuberculosis (3).

Mouse and rat TNF-α are predicted to be glycosylated, while human TNF-α is not (4).

  1. Aggarwal, B.B. (2003) Nat Rev Immunol 3, 745-56.
  2. Hehlgans, T. and Pfeffer, K. (2005) Immunology 115, 1-20.
  3. Lin, P.L. et al. (2007) J Investig Dermatol Symp Proc 12, 22-5.
  4. Pennica, D. et al. (1985) Proc Natl Acad Sci U S A 82, 6060-4.

Application References

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Companion Products

For Research Use Only. Not For Use In Diagnostic Procedures.

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