|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||Prohibitin Rabbit Recombinant mAb detects endogenous level of prohibitin.|
|Background||Human eukaryotic prohibitin (prohibitin-1 and prohibitin-2) is a membrane protein with different cellular localizations. It is involved in multiple cellular functions, including energy metabolism, proliferation, apoptosis, and senescence. The subcellular localization of prohibitin may determine its functions. Membrane prohibitin regulate the cellular signaling of membrane transport, nuclear prohibitin control transcription activation and the cell cycle, and mitochondrial prohibitin complex stabilize the mitochondrial genome and modulate mitochondrial dynamics, mitochondrial morphology, mitochondrial biogenesis, and the mitochondrial intrinsic apoptotic pathway. Prohibitin can also translocates into the nucleus or the mitochondria under apoptotic signals and the subcellular shuttling of prohibitin is necessary for apoptosis process. Any alteration in the content, post-transcriptional modification (i.e. phosphorylation) or the nuclear or mitochondrial translocation of prohibitin may influence cell fate.|