|Reactivity||Human Mouse Rat|
|Storage buffer||10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide.|
|Storage||Store at –20°C.|
1. Aspirate media from cultures and Wash the cells with 1X PBS. 2. Lyse cells by adding 1X SDS sample buffer and transfer the extract to a microcentrifuge tube. Keep onice. 3. Sonicate for 10–15 sec to complete cell lysis and shear DNA. 4. Heat a 20 µl sample to 95–100°C for 5 min, then cool on ice. 5. Centrifuge for 5 min (with Microcentrifuge). 6. Load appropriate volumes of samples onto SDS-PAGE gel (loading quantity of protein sample depends on the concentration of extracted proteins). NOTE: At the same time, please load the pre-stained molecular weight markers to determine molecular weights and verify electrotransfer. 7. Electrotransfer to nitrocellulose/PVDF membrane.
Membrane Blocking and Antibody Incubations
1. (Optional) After transfer, wash the transferred membrane with TBS for 5 min at room temperature. 2. Incubate the membrane in the blocking buffer for 1 hr at room temperature. 3. Wash three times for 5 min each with TBST.
b. Antibodies Incubation
1. Incubate membrane and primary antibody (at the appropriate dilution and diluent recommended) in a primary antibody dilution buffer with gentle agitation overnight at 4°C. 2. Wash three times for 5 min each with TBST. 3. Incubate membrane with an appropriate second antibodydissolved in the blocking buffer with gentle agitation for 1 hr at room temperature. 4. Wash three times for 5 min each with TBST. 5. Proceed with detection.
Detection of Proteins
1. After antibodies incubation, Wash membrane three times for 5 minutes in TBST. 2. PrepareECL Reagent (or other chromogenic agents/substrate according to your second antibody). Mix well. 3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.
Specimen Preparation (forcultured cell lines, IF-IC)
1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.
1. Add theblocking buffer and incubate for 60 min at RT.
|Specificity||ADAM17 Rabbit Recombinant mAb detects endogenous level of total ADAM17.|
|Background||A disintegrin and metalloproteinase 17 (ADAM17) is a major sheddase of membrane-anchored cytokines, cell adhesion molecules, receptors, ligands, and enzymes, such as tumor necrosis factor-α (TNF-α), transforming growth factor-α, TNF-α receptor 1 (TNFR1), TNF-α receptor 2 (TNFR2), and interleukin-6 receptor, L-selectin, Notch, and angiotensin-converting enzyme type 2, in which way ADAM17 could modulate a variety of signaling transduction and affect cell behavior. ADAM17 is regarded as a first line of defense against injury and infection, by releasing tumor necrosis factor α (TNFα) to promote inflammation and epidermal growth factor (EGF) receptor ligands to maintain epidermal barrier function. It is rapidly activated by a great variety of stimuli to induce the shedding and release of soluble TNFα and the EGFR ligands amphiregulin, epiregulin, heparin-binding EGF-like growth factor, and transforming growth factor α (TGFα). ADAM17 appears to have at least as many substrates as ADAM10 which include Notch cell fate regulators, amyloid precursor protein (APP), cellular prion protein, epidermal growth factor receptor (EGFR) ligands betacellulin and EGF, adhesion molecules E-cadherin, N-cadherin, VE-cadherin and CD44, transmembrane chemokines CX3CL1 and CXCL16, the low-affinity immunoglobulin E receptor CD23, and vascular endothelial growth factor receptor 2. Other ADAM17 substrates include the adhesion molecules ICAM-1, L-selectin, and TNF receptor family members TNFRI, TNFRII.|