Tumor necrosis factor alfa (TNF alfa) treatment to different cells rapidly and profoundly induced various proteins including zinc finger protein called A20 also known as tumor necrosis factor alfa-inducible protein 3(1). The function of A20 is to restrict and terminate inflammatory responses through modulation of the ubiqutination status of central components in NF-kB, interferon regulatory factor 3, IRF3 and apoptosis signal cascades (2). A20 tightly regulates NF-κB signaling by acting as an Ubiquitin editor, removing K63-linked ubiquitin chains and mediating addition of Ub chains that target substrates for degradation. In response to pro inflammatory cytokines, the deubiquitinase A20 interacts with regulatory molecules TAX1BP1, Itch and RNF to form the A20 editable ubiquitin-editing complex. The kinase IKKa and not IKKb is responsible for phosphorylation of TAX1BP1 at Ser593/Ser624 and the phosphorylation of TAXBP1 is pivotal for interaction of A20, Itch, RNF11 and phosphorylated TAXBP1 and down-regulation of signaling by NFkB.
Various polymorphisms with in the A20 locus resulted in multiple inflammatory and auto immune disorders including systemic lupus erythaematosis, rheumatoid arthritis, Crohn’s disease and psoriasis. A20 is also implicated in tumor suppression in several subsets of B-cell lymphomas (3). The NFkB inhibitory protein A20 significantly improves the recovery of liver function and mass following extended liver resection by optimizing energy production thru improved fatty acid metabolism and reduced inflammation (4). A20 undergoes specific glucose-triggered post-translational modifications (PTM) including O-GlcNAcylation and ubiquitination, which leads to its loss by proteasomal degradation, hence depriving the organism of a key element of its atheroprotective armamentarium and accelerating development of atherosclerotic lesions (5). A20 is a 790 aa (91kDa) protein and expressed in two isoform variants (A20 isoform a and isoform b).
The A20-selective antibodies were generated against unique antigenic sequence near C-terminal end (from amino acids 690-790). The antibodies to A20 are affinity purified over immobilized antigen based chromatography, and the purified immunoglobulins are stabilized in antibody stabilization buffer. The affinity purified antibodies are also conjugated to FITC and biotin for direct applications in IHC and cell sorting experiments. FabGennix Int. Inc., also provide limited quantities of antigenic blocking peptides for A20-101AP. Antibodies to A20 (A20-101AP) will label A20 protein in Western blot positive control sample and in many other cells. Limited quantities of Western blot positive control for A20 (PC-A20) and antigenic blocking peptide (P-A20) are available. Please inquire for their availability.
Immunogen: Synthetic peptide taken form near the C-terminal region of the A20 sequence from with in 690 and 790aa, the peptide was post-synthetically modified and conjugated to carrier protein to achieve desired antigenicity. The selected A20 peptide sequence was conserved in human, chimpenze, gaboon.
Concentration: A20-101AP and A20-FITC: IgG concentration 0.56-0.62mg/ml in antibody stabilization buffer.
Applications: Antibody A20-101AP is ideal for WB and ELISA applications, other applications have not been tested, but this will o. This antibody does not cross react to other calcium binding proteins. The species cross reactivity for this antibody is not examined fully. The dilutions for this antibody is for reference only, investigators are expected to determine the optimal conditions for specific assay. WB: > 1:500; IMM & i.p pull-down assays: n.d; IHC n.d. ELISA <1:10,000
Reactivity: This antibody detects a single band of approximately 91kDa in PC-A20 samples. When indicated n.d for specific application for this antibody does not means the antibody is not suitable for that application but rather has not yet tried for that application. Investigators using this antibody in applications not listed here can ask for a free complimentary sample.
Protocols: Standard protocol for various applications (WB; IMM and IHC) of this antibody can be requested by calling Technical support line, basic information on this antibody is provided with the product specification sheet, however, FabGennix Inc., strongly recommends investigators to optimize conditions for use of this antibody in their laboratories.
Form/Storage: The affinity-purified antibodies are supplied as stabilized product. Store at -20oC for long-term storage. FabGennix Inc. does not recommend storage of very dilute antibody solutions unless they are prepared in specially formulated multi use antibody dilution buffer (Cat # DiluOBuffer). Working solutions of antibodies in DiluOBuffer should be filtered through 0.45u filter after every use for long-term storage.
