Western blot analysis of I-kappa-B-alpha [pS32] was performed by loading 20 µg of HeLa (lane1) and Cos-7 (lane2) cell lysates 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. I-kappa-B-alpha [pS32] was detected at ~42 kDa using ABfinity™ I-kappa-B-alpha [pS32] Recombinant Rabbit Oligoclonal Antibody (7101295) at 1-3 µg/mL in 2.5 % skim milk at 4ºC overnight on a rocking platform. To confirm specificity, the corresponding blot on right was incubated with lambda phosphatase and its reactivity with antibody was tested. 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, Non-human primate|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Peptide 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)||1-3µg/10^6 cells|
|Western Blot (WB)||1-3µg/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 mouse, rabbit and rat 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™ oligoclonal antibodies comprise a selection of multiple different recombinant monoclonal antibodies, providing the best of both worlds—the sensitivity of a polyclonal antibody with the specificity of a monoclonal, all delivered with the consistency only found in a recombinant antibody. While functionally the same as a polyclonal antibody—recognizing multiple epitope sites on the target and producing higher detection sensitivity for low abundance targets when compared with monoclonal antibodies—an oligoclonal antibody has a known mixture of light and heavy chains. This exact population can be produced in every lot, circumventing the biological variability typically associated with polyclonal antibody production.
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.