|Reactivity||Human Mouse Rat|
|MW (kDa)||10, 90kDa|
|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||NEDD8 Rabbit Recombinant mAb detects endogenous level of total NEDD8.|
|Background||NEDD8 is a ubiquitin-like modifier most well-studied for its role in activating the largest family of ubiquitin E3 ligases, the cullin-RING ligases (CRLs). Conjugation of the ubiquitin-like (Ubl) protein NEDD8 (neural precursor cell expressed developmentally downregulated protein 8) to target proteins, or neddylation, is a fundamental biological process that controls many key cellular functions, including cell cycle progression, the DNA damage response, and apoptosis. The most well-studied target for NEDD8 conjugation are the cullins (CUL1,−2,–3,−4A,−4B,−5, and −7), which are the scaffold subunits of the largest family of ubiquitin E3 ligases, the cullin-RING ligases (CRLs). NEDD8 transfer from the E2 to a lysine (Lys) residue within the cullin promotes conformational changes that increase the catalytic activity of the CRL, while also blocking the binding of the CRL exchange factor CAND1. Structural changes induced by NEDD8 conjugation to cullin subunits ultimately contribute to the efficient ubiquitylation of downstream CRL substrates and their degradation by the 26S proteasome. Several non-cullin neddylation targets have been discovered: p53, E2F1, ribosomal protein L11, Smurf1 and Histone H4 and so on.|