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Western blot analysis of I-kappa-B-alpha [pS32] was performed by loading 30 µg of THP-1 (lane1), Jurkat (lane2), Jurkat treated for 60 minutes with 50 µg/mL of TNF alpha (lane3), HeLa (lane4), HeLa treated for 60 minutes with 50 µg/mL of TNF alpha (lane5) and HeLa treated for 60 minutes with 50 µg/mL of TGF Beta 1 (lane6) cell lysate using NuPAGE® Novex® 10% Bis-Tris gel (NP0301BOX), 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. I-kappa-B-alpha [pS32] was detected at ~42 kDa using ABfinity™ I-kappa-B-alpha [pS32] Recombinant Rabbit Monoclonal Antibody (701271) at 2-3 µg/mL in 2.5% skim milk at 4°C overnight on a rocking platform. 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||Phosphopeptide corresponding to amino acids 27–36 of human I K B-Alpha [pS32]|
|Contains||0.09% sodium azide|
|Storage Conditions||Maintain refrigerated at 2-8°C for up to 1 month. For long term storage store at -20°C|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||3-5 µg/10^6 cells|
|Western Blot (WB)||1-3 ug/ml|
* 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.
This antibody is predicted to react with equine, rabbit and non-human primate based on sequence homology.
ABfinity™ recombinant antibodies are rabbit monoclonal antibodies, unmatched for producing superior results. ABfinity™ antibodies are developed by immunizing animals, screening for functionality, cloning the immunogen-specific antibody genes into high-level mammalian expression vectors, produced on a large scale, and purified with Protein A.
ABfinity™ monoclonal antibodies resemble rabbit monoclonals isolated from serum or produced by hybridomas, but demonstrate greater specificity and sensitivity. Because ABfinity™ recombinant antibodies are derived from cloned DNA sequences of the heavy and light antibody chains, they are not susceptible to cell-line drift or lot-to-lot variation, thus allowing for peak specificity and performance.
Intact IgG appears on a non-reducing gel as ~150 kDa band and upon reduction generating a ~25 kDa light chain band and a ~50 kDa heavy chain.
I kappa B-alpha is a 40 kDa protein that functions to inhibit NF- kappaB activity. The inhibition occurs via protein-protein interaction between I kappaB proteins and NF- kappaB dimers in the cytosol. The interaction of I kappa B-alpha with NF- kappaB masks the nuclear localization sequence of NF- kappaB, preventing NF- kappaB translocation to the nucleus. A variety of stimuli can activate gene expression by liberating NF- kappaB through the degradation of I kappaB alpha. These stimuli include the proinflammatory cytokines TNF- alpha and IL-1 beta, chemokines, PMA, growth factors, LPS, UV irradiation, viral infection, as well as various chemical and physical stresses. In humans, the gene is located on the q arm of chromosome 14.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
I-kappa-B-alpha; IkappaBalpha; IkB-alpha; IKBA; MAD-3; Major histocompatibility complex enhancer-binding protein MAD3; NF-kappa-B inhibitor alpha; NFKBI; nuclear factor of kappa light chain gene enhancer in B-cells; nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha
IKBA; MAD-3; MAD3; NFKBI; NFKBIA