|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||IFNAR1 Rabbit Recombinant mAb detects endogenous level of total IFNAR1.|
|Background||The interferon-α/β receptor (IFNAR) is a virtually ubiquitous membrane receptor which binds endogenous type I interferon (IFN) cytokines. Type I IFN receptor forms a ternary complex, composed of its two subunits IFNAR1 and IFNAR2, and a type I IFN ligand. IFNAR1 is the low affinity subunit. Its cytoplasmic domain associates with Tyk2 and Tyk2 is required for membrane expression of IFNAR1. In the absence of Tyk2, a cytoplasmic IFNAR1 motif is phosphorylated, inducing receptor internalization. IFNAR1 then localizes to the perinuclear endosomal compartment and is degraded. IFNAR1 binding to low affinity ligands, such as IFNα subtypes, has been proposed to be the rate limiting step in the ternary complex formation.|