|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.
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.
1. Add theblocking buffer and incubate for 60 min at RT.
|Specificity||IRF1 Rabbit Recombinant mAb detects endogenous level of total IRF1.|
|Background||Interferon Regulatory Factor 1 (IRF1) is an essential player in many facets of the immune response and oncogenesis. The IRF1 protein is also very unstable (half life ~30 min) compared with IRF2 (half-life ~8 hrs). IRF1 is expressed at low levels in unstimulated cells and is activated by many cytokines including type I (IFN , IFN , and others) and II (IFN ) interferons, tumor necrosis factor- (TNF- ), retinoic acid, interleukin-1 (IL-1), IL-6, and viral infection. Initial signaling is mediated through the Janus-activated kinase-signal transducer and activator of transcription (JAK/STAT1) pathway, leading to the activation of the IRF1 promoter by the Stat and NF-κB transcription factors. When a signal is transduced through the IFN receptor, phosphorylated STAT1 translocates to the nucleus where it induces the transcription of primary IFNγ response genes . Stat1-deficient cells no longer respond to IFN stimulation by inducing IRF1 expression. IRF1 has remarkable functional diversity and controls the transcription of genes involved in mediating antiviral, immunomodulatory, and antiproliferative effects, also involved in cell cycle regulation and apoptosis in response to a variety of stressors.|