BID antibody (AA 61-118)

Catalog No. ABIN5540048

This Mouse Monoclonal antibody specifically detects BID in WB. It exhibits reactivity toward Human.

Quick Overview for BID antibody (AA 61-118) (ABIN5540048)

Target: BID (BH3 Interacting Domain Death Agonist (BID))

Reactivity: Human

Host: Mouse

Clonality: Monoclonal

Conjugate: This BID antibody is un-conjugated

Application: Western Blotting (WB)

Clone: 5C9

Product Details anti-BID Antibody

Binding Specificity: AA 61-118

Specificity: This antibody reacts with BID.

Cross-Reactivity (Details): Does not work with Mouse (WR19L, L5178Y, NIH/3T3) and Rat (Rat-1 BHK).

Purification: Protein A agarose

Immunogen: Recombinant human BID (61-118 aa)

Isotype: IgG1

Application Details

Application Notes: Western blot: 1-5 μg/mL for chemiluminescence detection system. 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 cold 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 1:10,000 HRP-conjugated anti-mouse 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 chemilumi nescence 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 ZR-75-1, U937, HL-60)

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 BID

Target: BID (BH3 Interacting Domain Death Agonist (BID))

Alternative Name: bid

Background: Apoptosis is a major form of cell death characterized by several morphological features that include chromatin condensation and fragmentation, cell membrane blebbing, and formation of apoptotic bodies. BID is an apoptosis promoting protein, which possesses only BH3 domain that is a common structure of Bcl-2 family members. Once full length BID (p22) is cleaved with caspases, cleaved BID (p 15) induces a conformational change and oligomerization of a protein named BAK on mitochondrial membrane. It is thought that oligomerized BAK contribute to forming pore s that release cytochrome c into cytosol.

UniProt: P55957

Pathways: Apoptosis, Caspase Cascade in Apoptosis, Positive Regulation of Endopeptidase Activity