|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||Phospho-Nrf2 (S40) Rabbit Recombinant mAb detects endogenous level of total Phospho-Nrf2 (S40).|
|Background||Nrf2 is the key transcription factor regulating the antioxidant response. Nrf2 signaling is regulated by compartmental segregation. Under basal conditions, Nrf2 is found mainly sequestered in the cytoplasm. When challenged by oxidative stress derived from accumulation of ROS or reactive nitrogen species (RNS), Nrf2 can quickly translocate into the nucleus and elicit the antioxidant response. At least four components in combine, namely Nrf2, Keap1 (Kelch-like ECH-associated protein 1), a group of small musculoaponeurotic fibrosarcoma (Maf) proteins and a cis-acting enhancer called antioxidant response element (ARE) or electrophile responsive element (EpRE), are essential for the antioxidant response. Nrf2 signaling is repressed by Keap1 at basal condition and induced by oxidative stress. PKC phosphorylation of the Serine-40 (S40) residue of Nrf2 was reported to facilitate Keap1/Nrf2 dissociation and Nrf2 nuclear translocation.|