|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||Phospho-RSK1 (S380) Rabbit Recombinant mAb detects endogenous level of total phospho-RSK1 (S380).|
|Background||Ribosomal S6 kinase 1 (RSK1) belongs to a family of proteins with two kinase domains. Following activation in the cytoplasm by extracellular signal-regulated kinases (ERK1/2), it mediates the cell-proliferative, cell-growth, and survival-promoting actions of a number of growth factors and other agonists. Among the four isoforms of RSKs that are products of different genes, RSK1, RSK2, and RSK3 share significant sequence similarity; RSK4 is longer than the other isoforms and may also be functionally different. Despite their similarity, RSK1, RSK2, and RSK3 are not functionally redundant. The activation of RSK1 depends on the docking of ERK1/2 to the C-terminus. Following docking onto RSK1, ERK1/2 phosphorylates T573 in the activation loop of the C-terminal kinase (CTK), as well as T359 and S363 in the linker region that joins the N-terminal kinase (NTK) and CTK. Phosphorylation of T573 activates the CTK, and this domain can then phosphorylate S380 in the linker region. Phosphorylation of S380 provides a docking site for phosphoinositide-dependent kinase 1 (PDK1), which can then phosphorylate S221 in the activation loop of the NTK and permits RSK1 to phosphorylate its downstream substrates. On activation of RSK1 in the cytoplasm and at the plasma membrane, the fully active RSK1 localizes to the nucleus.|