|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||JunB Rabbit Recombinant mAb detects endogenous level of total JunB.|
|Background||The AP-1 transcription factor participates in the control of cellular responses to stimuli that regulate proliferation, differentiation, immune response, cell death and the response to genotoxic agents or stress. AP-1 is composed of Jun family members (c-Jun, JunB, and JunD) that can form either homo- or hetero-dimers among themselves. Jun proteins also dimerize with Fos family members (c-Fos, FosB, Fra1 and Fra2) Jun and Fos proteins also dimerize efficiently with other transcription factors such as members of the ATF/CREB or Maf/Nrl families of proteins. The JunB protein contains a JNK docking site but lacks N-terminal acceptor serine residues. While c-Jun is a strong transcriptional activator of cyclin D1, JunD is a weak positive activator and JunB even an inhibitor of this promoter. JunB is also a Jun member that antagonizes the Ras-signalling pathway. JunB differs in biological properties from its homologue c-Jun and was defined as a negative regulator of cell growth.|