|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-p53 (S392) Rabbit Recombinant mAb detects endogenous levels of total phospho-p53 (S392).|
|Background||p53 is a crucial tumor suppressor, long-recognized to suppress cancer through the induction of cell-cycle-arrest or apoptosis programs in response to a plethora of different cellular stress signals. Post-translational modifications play important roles during the stabilisation and activation of p53 by various genotoxic and non-genotoxic stresses. The highly conserved residue, Ser392 (Ser389 in mice), is a major phosphorylation site in p53. Ser392 is phosphorylated under normal, unstimulated conditions but the level of this phosphorylation is increased significantly in response to a range of stimuli each of which leads to p53 stabilisation. Ser392 has been reported to be a major UV-stimulated phosphorylation site that is modified through the p38 MAPK pathway in a manner that may involve recruitment of CK2. Phosphorylation of Ser392 is an integral event that occurs not only in response to UV, but also during the induction of p53 by a range of stimuli including treatment of cells with the MDM2 inhibitor, Nutlin 3a.|