TRAF6 antibody

Catalog No. ABIN5541395

This Hamster Monoclonal antibody specifically detects TRAF6 in WB and IP. It exhibits reactivity toward Mouse.

Quick Overview for TRAF6 antibody (ABIN5541395)

Target: TRAF6 (TNF Receptor-Associated Factor 6 (TRAF6))

Reactivity: Mouse

Host: Hamster

Clonality: Monoclonal

Conjugate: This TRAF6 antibody is un-conjugated

Application: Western Blotting (WB), Immunoprecipitation (IP)

Clone: 1F8

Product Details anti-TRAF6 Antibody

Specificity: This antibody reacts with TRAF6 (60 kDa).

Purification: Protein G agarose

Immunogen: Recombinant mouse TRAF6

Isotype: IgG

Application Details

Application Notes: Western blot: 1 μg/200 mL for chemiluminescence detection system. Immunoprecipitation: 1 μg/200 μL of cell extract from 5x10 6 cells. For details see protocol below.

Protocol: SDS-PAGE & Western blotting 1) Wash the cells 3 times with PBS and suspend with 10 volume of cold Lysis buffer (50 mM Tris-HCl, pH 7.2, 250 mM NaCl, 0.1 % NP-40, 2 mM EDTA, 10 % glycerol) containing appropriate protease inhibitors. Incubate it at 4 o C with rotating for 30 minutes, then sonicate briefly (up to 10 seconds). 2) Centrifuge the tube at 12,000 x g for 10 minutes at 4 o C and transfer the supernatant to another tube. Measure the protein concentration of the supernatant and add the Lysis buffer to make 8 mg/mL solution. 3) Mix the sample with equal volume of Laemmli's sample buffer. 4) Boil the samples for 2 minutes and centrifuge. Load 10 μ L of the sample per lane in a 1 mm thick SDS-polyacrylamide gel for electrophoresis. 5) Blot the protein to a polyvinylidene difluoride (PVDF) membrane at 1 mA/cm 2 for 1 hour in a semi-dry transfer system (Transfer Buffer: 25 mM Tris, 190 mM glycine, 20 % MeOH). See the manufacture's manual for precise transfer procedure. 6) To reduce nonspecific binding, soak the membrane in 10 % skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature, or overnight at 4 o C. 7) Incubate the membrane with primary antibody diluted with PBS, pH 7.2 containing 1 % skimmed milk as suggest in the APPLICATIONS for 1 hour at room temperature. (The concentration of antibody will depend on condition.) 8) Wash the membrane with PBS-T [0.05 % Tween-20 in PBS] (5 minutes x 6 times). 9) Incubate the membrane with the 1:1,000 HRP-conjugated anti-Hamster IgG diluted with 1 % skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature. 10) Wash the membrane with PBS-T (5 minutes x 6 times). 11) Wipe excess buffer on the membrane, then incubate it with appropriate chemiluminescence reagent for 1 minute. Remove extra reagent from the membrane by dabbing with paper towel, and seal it in plastic wrap. 12) Expose to an X-ray film in a dark room for 5 minutes. Develop the film as usual. The condition for exposure and development may vary. (Positive controls for Western blotting L5178Y, WR19L, NIH/3T3) Immunoprecipitation 1) Wash the cells 3 times with PBS and suspend with 10 volume of cold Lysis buffer (50 mM Tris-HCl pH 7.2, 250 mM NaCl, 0.1 % NP-40, 2 mM EDTA, 10 % glycerol) containing appropriate protease inhibitors. Incubate it at 4 o C with rotating for 30 minutes, then sonicate briefly (up to 10 seconds). 2) Centrifuge the tube at 12,000 x g for 10 minutes at 4 o C and transfer the supernatant to another tube. 3) Add primary antibody as suggest in the APPLICATIONS into 200 μ L of the supernatant. Mix well and incubate with gentle agitation for 30-120 minutes at 4 o C. Add 20 μ L of 50 % protein G agarose beads resuspended in the cold Lysis buffer. Mix well and incubate with gentle agitation for 60 minutes at 4 o C. 4) Wash the beads 3-5 times w ith the cold Lysis buffer (centrifuge the tube at 2,500 x g for 10 seconds). 5) Resuspend the beads in 20 μL of Laemmli's sample buffer, boil for 3-5 minutes, and centrifuge for 5 minutes. Use 10 μL/lane for the SDS-PAGE analysis. (See SDS-PAGE & Western blotting.) (Positive control for Immunoprecipitation L5178Y)

Restrictions: For Research Use only

Handling

Format: Liquid

Buffer: PBS containing 50 % glycerol, pH 7.2. No preservative is contained.

Preservative: Azide free

Storage: -20 °C

Storage Comment: Upon receipt, store undiluted (in aliquots) at -20°C. Avoid repeated freezing and thawing. Shelf life: One year from despatch.

Target Details for TRAF6

Target: TRAF6 (TNF Receptor-Associated Factor 6 (TRAF6))

Alternative Name: traf6,rnf85

Background: TRAFs (TNF receptor associated factors) form a family of cyt oplasmic adaptor proteins that mediate signal transduction from many members of the TNF-receptor superfamily. TR AF6 also participates in signal transduction of the IL-1R/Toll receptor superfamily. TRAF6 is a ~55 kDa cytoplasmi c adapter protein that plays diverse roles in perinatal and postnatal survival, bone metabolism, adaptive immunity, cytokine signaling, and the regulation of inflammatory and apoptotic responses through activation of the transcription factors of NF- κ B. TRAF6 overexpression activates MAP kinases, including ERK, p38 and JNK, and TRANCE/OPGL Akt/PKB through a signaling complex involving c-Src and TRAF6. TRAF6 dimerizes strongly with itself and weakly with TRAF2 and TRAF3.

UniProt: P70196

Pathways: NF-kappaB Signaling, TCR Signaling, TLR Signaling, Fc-epsilon Receptor Signaling Pathway, Neurotrophin Signaling Pathway, Activation of Innate immune Response, Regulation of Leukocyte Mediated Immunity, Positive Regulation of Immune Effector Process, Production of Molecular Mediator of Immune Response, Tube Formation, Hepatitis C, Toll-Like Receptors Cascades, Ubiquitin Proteasome Pathway