Immunofluorescence analysis of Phospho-IKK-alpha pSer176 / pSer180 was done on 70% confluent log phase HeLa cells. The cells were treated for 20 minutes with 50ng/ml of TNF-Alpha and fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with Phospho-IKK-alpha pSer176 / pSer180 Rabbit Polyclonal Antibody (44714) at 1ug/mL in 1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d is a merged image showing Nuclear localization. Panel e is a no primary antibody control. The images were captured at 60X magnification.
|Tested species reactivity||Human|
|Host / Isotype||Rabbit / IgG|
|Immunogen||The antiserum was produced against a chemically synthesized phosphopeptide derived from the region of human IKK-alpha that contains serine 176 and serine 180.|
|Storage buffer||Dulbecco's PBS, pH 7.3, with 1mg/ml BSA|
|Contains||0.05% sodium azide|
|Tested Applications||Dilution *|
|ChIP assay (ChIP)||10µl|
|Flow Cytometry (Flow)||3-5 µg/million cells|
|Immunocytochemistry (ICC)||1 µg/ml|
|Immunofluorescence (IF)||1 µg/ml|
|Western Blot (WB)||0.1-1.0 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.
Nuclear factor kappa B (NF-kappa-B) is a ubiquitous transcription factor and an essential mediator of gene expression during activation of immune and inflammatory responses. NF-kappa-B mediates the expression of a great variety of genes in response to extracellular stimuli including IL-1, TNF-a and bacteria product LPS. NF-kappa-B is associated with Ikappa-B proteins in the cell cytoplasm, which inhibit NF-kappa-B activity. The long-sought Ikappa-B kinase (IKK), which phosphorylates Ikappa-B, and mediates Ikappa-B degradation and NF-kappa-B activation, was recently identified by several laboratories. IKK is a serine protein kinase, and the IKK complex contains alpha and beta subunits (IKK-a and IKK-b). IKK-a and IKK-b interact with each other and both are essential for the NF-kappa-B activation. IKK-a specifically phosphorylates Ikappa-B-alpha. IKK-a is expressed in a variety of human tissues.
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