|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.
|Specificity||ITCH Rabbit Recombinant mAb detects endogenous level of total ITCH.|
|Background||Itch is a monomeric protein, which belongs to the homologous to E6-AP carboxy terminus (HECT)-type family of E3s, whose modular structural organization consists of an N-terminal Ca2+-dependent phospholipid-binding C2 domain, multiple protein-protein interaction WW domains, and a C-terminal HECT domain. Although a small fraction of Itch displays a perinuclear distribution overlapping with the trans-Golgi network, the protein is predominantly associated with early and late endosomal compartments and lysosomes. Localization of Itch to the endocytic vesicles is mediated by the C2 domain. Itch typically regulates the stability of both transmembrane receptors through canonical monoubiquitylation or multiubiquitylation, and intracellular substrates through polyubiquitylation, driving them to lysosomal and proteasomal degradation, respectively. Proteolysis-independent ubiquitylation events have been also ascribed to the E3 activity of Itch. The ubiquitin ligase Itch plays key roles in different cellular contexts, in virtue of its functionally distinct substrates. A significant number of the transcriptional regulators targeted by Itch for proteasomal destruction are crucially involved in controlling cell growth, differentiation, and apoptotic processes.|