|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||Ret Rabbit Recombinant mAb detects endogenous level of total Ret.|
|Background||The RET proto-oncogene encodes a receptor tyrosine kinase with four cadherin-related motifs and a cysteine-rich region in the extra-cellular domain, and its ligands are glial cell line-derived neurotrophic factor (GDNF) and related molecules, includingneurturin (NRTN), artemin (ARTN) and persephin (PSPN). RET activation by these neurotrophic factors is mediated through aunique multi-component receptor system, consisting of glycosyl-phosphatidylinositol-anchored coreceptor (GFRα1-4) as a ligand-binding component and RET tyrosine kinase as a signaling component. The intracellular domain of RET contains autophosphorylation sites, and phosphorylated tyrosines serve as docking sites for signaling molecules. Like the majority of receptor tyrosine kinases studied, signaling pathways initiated by the RET receptor include the Ras/MAP kinase, PI3 kinase/AKT, and phospholipase C-γ (PLCγ) pathways. In addition to tyrosine autophosphorylation, RET has been found to undergo serine phosphorylation at Ser696 by protein kinase A (PKA).|