Immunofluorescence analysis of NF-kappa-B p65 was done on 70% confluent log phase TNF-Alpha treated HeLa cells (serum starved for 16 hours followed by treatment with 20 ng/ml TNF-Alpha for 1 hour). The cells were fixed with 4% paraformaldehyde for 15 minutes; permeabilized with 0.25% Triton X-100 for 10 minutes followed by blocking with 5% BSA for 1 hour at room temperature. The cells were incubated with ABfinity NF-kappa-B p65 Recombinant Rabbit Oligoclonal Antibody (710048) at 1µg/mL in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Flour 488 Goat Anti-Rabbit IgG Secondary Antibody (A11008) at a dilution of 1:400 for 30 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 594 Phalloidin (A12381). Panel d shows nuclear localization of SMAD2 upon treatment; Panel e showing cytoplasmic localization in untreated HeLa cells. Panel f is a no primary antibody control. The images were captured at 20X magnification.
|Tested species reactivity||Human, Mouse|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Recombinant protein corresponding to amino acids 375-551 of human NF kappaB p65.|
|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 *|
|ChIP assay (ChIP)||3µg|
|Immunohistochemistry (Paraffin) (IHC (P))||1:10-1:100|
|Western Blot (WB)||0.5-1µg/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.
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.
NF kappaB p65 belongs to the Rel family of transcription factors. NF kappaB homo and heterodimers are retained in an inert, non-DNA-binding form in the cytosol by inhibitory molecules in most cells. Activation of NF kappaB occurs predominantly through the phosphorylation and degradation of I kappaB proteins. The free NF kappaB translocates to the nucleus, where it upregulates expression from genes involved in developmental processes, cellular growth, apoptosis, and immune and inflammatory responses. In humans, the gene is located on the q arm of chromosome 11.
IP-MS enrichment of RELA (LFQ intensity): RELA was enriched 282-fold from A549 lysate compared to background proteins, using the optimized IP-MS workflow with Pierce MS-Compatible Magnetic IP Kit protein A/G (Part No. 90409) and RELA antibody (Part No. 710048). The STRING database (www.string-db.org) was used to identify the protein interactor list. See more information on IP-MS verification of antibody selectivity. IP-MS validation info.
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