|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||STAT6 Rabbit Recombinant mAb detects endogenous levels of total STAT6.|
|Background||Signal transducer and activator of transcription (STAT) proteins are critical mediators of cytokine signaling. Among the seven STAT proteins, STAT6 is activated by IL-4 and IL-13 and plays a predominant role in the immune system. Once these cytokines bind to their cognate receptors, the associated Janus Kinases (Jak) are activated and phosphorylate conserved tyrosine residues on the receptor. Latent cytoplasmic STAT6 docks onto the phosphorylated receptor via an SH2 domain allowing the Jaks to phosphorylate the conserved tyrosine-641 on STAT6. Once phosphorylated, STAT6 forms homodimers and translocates to the nucleus. In the nucleus, STAT6 has the ability to bind directly to DNA via a DNA-binding domain and is able to regulate transcription. IL-4, IL-13, and STAT6 promote humoral immunity, clearance of helminthic parasites as well as the pathogenesis of allergic disorders like asthma, food allergies, and atopic dermatitis. STAT6 is critical for a number of responses in T cells, including the development of T-helper type 2 (Th2) cells and IL-4-stimulated proliferative responses. In B cells, STAT6 promotes immunoglobulin class switching to IgE and IgG1 and the expression of some of the cell surface molecules responsible for antigen presentation by B cells. In addition to a requirement in T and B cells, STAT6 also functions in macrophages and dendritic cells. In macrophages, STAT6 promotes IL-4-induced differentiation of alternatively activated macrophages (AAM) and mediates IL-13-induced expression of genes such as MHC class II. STAT6 also functions in other tissue, including mammary gland, lung, and skin.|