Western blot analysis of p50 [pS337] was performed by loading 20 µg of Jurkat, Jurkat treated with TNF-alpha (20 ng/mL for 30 minutes) lysates (lane 1,2) using Novex® NuPAGE® 4-12% Bis-Tris gel (NP0321BOX), XCell SureLock™ Electrophoresis System (EI0002), Novex® Sharp Pre-Stained Protein Standard (LC5800), and iBlot® Dry Blotting System (IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5% skim milk for 1 hour at room temperature. p50 [pS337]was detected at ~50 kDa using ABfinity™ p50 [pS337] Recombinant Rabbit Monoclonal Antibody (701430) at 1:1000 dilution in 2.5% skim milk at 4°C overnight on a rocking platform. To confirm specificity, competition was performed with the phosphopeptide (10 µg/mL) (lane 3, 4). Goat Anti-Rabbit IgG-HRP Secondary Antibody (G21234) at 1:5000 dilution was used and chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (WP20005).
|Tested species reactivity||Human|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Phosphorylation of the p50 subunit at the Ser-337 residue|
|Contains||0.09% sodium azide|
|Storage Conditions||Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||Assay Dependent|
|Western Blot (WB)||1:500-1:2000|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
NF-kB is a nuclear transcription factor activated by various extra and intracellular stimuli such as cytokines, UV radiation, stress, in injury, and by bacterial and viral products. It is involved in regulation of various cellular events including cell growth, differentiation, proliferation, apoptosis and inflammation. NFKB1 (p50), a 50KDa functional sub-unit of NF-kB, is a member of Rel protein family. It is synthesized as a p105 precursor protein and consists of an N-terminal conserved RHD-region containing nuclear localization signal, DNA-binding and dimerization domains. NFKB1 (p50) forms homodimers or heterodimerizes with p65, forming the functional NF-kB factor. NFKB1 (p50) directs the nuclear translocation of NF-kB and is instrumental in its DNA-bidning. Pathological role of NF-kB has been suggested in AIDS, hematogenic cancer cell metastasis and rheumatoid arthritis.
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