Cat.No.:A5264
WB
Application | WB,ELISA | ||
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Dilution |
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Reactivity | Human Mouse Rat | ||
MW (kDa) | 90kDa | ||
Source | Rabbit | ||
Concentration | 1mg/ml | ||
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. |
WB | + Expand Western Blotting Sample preparation 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 a. Blocking 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. |
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IP | + Expand IP Sample preparation 1. To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold 1X PBS. Cell Lysate Pre-Clearing (Optional) Pre-clearing the lysate can help reduce non-specific binding and reduce background. 1. Vortex to mix beads. Immunoprecipitation 1. Add primary antibody at the appropriate dilutionto cell lysat. Incubate with gentle agitation or rotation overnight at 4°C. Sample Analysis a. Western Immunoblotting 1. Resuspend the pellet with SDS sample buffer. Vortex, then centrifuge for 30 sec at 14,000 x g. b. Kinase Assay 1. Wash the pellet twice with 500 µl 1X kinase buffer. Keep on the ice. |
IHC | + Expand Immunohistochemistry (Paraffin) Deparaffinization/Rehydration NOTE: Do not allow slides to dry at any time during this procedure. 1. Deparaffinize/hydrate sections: 1. Incubate sections in three washes of xylene for 5 min each. 2. Wash sections two times in dH2O for 5 min each. Antigen retrieval For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min. Staining 1. Wash sections in dH2O three times for 5 min each. |
IF | + Expand Immunofluorescence (Immunocytochemistry) 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. Immunostaining 1. Add theblocking buffer and incubate for 60 min at RT. |
FCM | + Expand Flow Cytometry Fixation NOTE: lyse red blood cells and wash by centrifugation prior to fixation if using whole blood. Permeabilization 1. Permeabilize the cells by adding ice-cold 100% methanol slowly, with gently vortexing.The final concentration of methanol is 90%. Immunostaining NOTE: Count the cells using a hemocytometer or an alternative method. E. Optional DNA Dye 1. Resuspendthe cells in DNA dye. |
CHIP | + Expand Chromatin IP Cell Culture Cross-linking and Sample Preparation For optimal ChIP results, use approximately 4 X 106 cells for each experiment. One additional sample should be processed for Analysis of Chromatin Digestion and Concentration. Include one extra dish of cells in experiment to be used for determination of cell number using a hemocytometer. Nuclei Preparation and Chromatin Digestion Before starting Analysis of Chromatin Digestion and Concentration 1. To the 50 µl chromatin sample, add 100 µl nuclease-free water, 6 µl 5 M NaCl, and 2 µl RNAse A. Vortex to mix and incubate at 37°C for 30 min. Chromatin Immunoprecipitation For optimal ChIP results, use approximately 5 to 10 µg of digested, cross-linked chromatin per IP reaction. Typically, 100 µl of digested chromatin is diluted into 400 µl 1X ChIP Buffer prior to the addition of antibodies. However, if more than 100 µl of chromatin is required per IP, the cross-linked chromatin preparation does not need to be diluted as described below. Antibodies can be added directly to the undiluted chromatin preparation for immunoprecipitation of chromatin complexes. Elution of Chromatin and Reversal of Cross-links Before starting DNA Purification This part was performed at room temperature Data analysis Purified DNA can be used in PCR (standard PCR or Real-Time Quantitative PCR). |
Specificity | Phospho-RSK1 (S380) Rabbit Recombinant mAb detects endogenous level of total phospho-RSK1 (S380). |
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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. |