Immunohistochemistry analysis of Phospho-NFkB [pS529] showing staining in the nucleus and cytoplasm of paraffin-embedded human spleen tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Phospho-NFkB [pS529] polyclonal antibody (44711G) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.
|Tested species reactivity||Human, Mouse|
|Host / Isotype||Rabbit / IgG|
|Immunogen||The antiserum was produced against a chemically synthesized phosphopeptide derived from a region of human NFkB that contains serine 529.|
|Purification||Antigen affinity chromatography|
|Storage buffer||Dulbecco's PBS, pH 7.3, with 1mg/ml BSA, 50% glycerol|
|Contains||0.05% sodium azide|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||3-5 µg/million cells|
|Immunohistochemistry (Paraffin) (IHC (P))||1:10-1:100|
|Western Blot (WB)||Assay Dependent|
* 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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