Nuclear Factor-KB P65 (NFkBP65) (AA 136-224) antibody

Details for Product No. ABIN968223
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Antigen
Synonyms c-Rel, C-Rel, Xrel2, c-rel, rel-A, v-rel, xrel, NFKB3, p65, NFkB, zgc:100833
Epitope
AA 136-224
(66), (57), (46), (46), (36), (34), (33), (31), (31), (30), (28), (20), (19), (12), (11), (7), (7), (7), (7), (6), (6), (6), (4), (4), (4), (3), (3), (3), (3), (3), (3), (3), (3), (2), (2), (2), (2), (2), (2), (2), (2), (2), (2), (2), (2), (2), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1)
Reactivity
Human
(853), (443), (352), (76), (75), (74), (38), (9), (8), (6), (2), (2), (1), (1)
Host
Mouse
(800), (65), (6), (5), (1)
Clonality (Clone)
Monoclonal ()
Conjugate
Un-conjugated
(24), (19), (15), (11), (10), (10), (10), (10), (10), (10), (10), (10), (10), (10), (1), (1), (1)
Application
Western Blotting (WB), BioImaging (BI)
(654), (335), (332), (170), (128), (121), (100), (38), (21), (18), (16), (12), (7), (5), (2), (1)
Pubmed 5 references available
Quantity 150 µg
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Catalog No. ABIN968223
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Immunogen Human NF-kappaB
Clone LEP-02
Isotype IgG1
Cross-Reactivity Dog (Canine), Frog, Rabbit, Rat (Rattus)
Characteristics 1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
2. Please refer to us for technical protocols.
3. This antibody has been developed and certified for the bioimaging application. However, a routine bioimaging test is not performed on every lot. Researchers are encouraged to titrate the reagent for optimal performance.
4. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
5. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
6. Triton is a trademark of the Dow Chemical Company.
Purification Purified from tissue culture supernatant or ascites by affinity chromatography.
Alternative Name NF-kappa B p65
Background NF-kappaB is a ubiquitously expressed transcription factor that regulates many cytokine and Ig genes. It is involved in immune, inflammatory, viral, and acute phase responses. The most studied NF-kappaB complex consists of the p50 and p65 subunits, both containing a 300 amino acid region with homology to the Rel proto-oncogene product. The p50 subunit binds DNA, whereas the p65 subunit is responsible for the interaction of NF-kappaB with its inhibitor, IkappaB. In most cell types, the p50/p65 heterodimer is located within the cytoplasm complexed to IkappaB. This complex prevents nuclear translocation and activity of NF-kappaB. In response to stimuli such as cytokines, LPS, and viral infections, IkappaB is phosphorylated at critical residues. This phosphorylation induces dissociation of the IkappaB/NF-kappaB complex, allowing the free heterodimeric NF-kappaB to form a heterotetramer that translocates to the nucleus. In the nucleus, it binds to the kappaB site within promoters and enhancers and functions as a transcriptional activator.
Molecular Weight 65 kDa
Application Notes Bioimaging
1. Seed the cells in appropriate culture medium at ~10,000 cells per well in an 96-well Imaging Plate and culture overnight.
2. Remove the culture medium from the wells, and fix the cells by adding 100 myl of Fixation Buffer to each well. Incubate for 10 minutes at room temperature (RT).
3. Remove the fixative from the wells, and permeabilize the cells using either 90% methanol, or Triton™ X-100: a. Add 100 myl of -20°C 90% methanol to each well and incubate for 5 minutes at RT. OR b. Add 100 myl of 0.1% Triton™ X-100 to each well and incubate for 5 minutes at RT.
4. Remove the permeabilization buffer, and wash the wells twice with 100 myl of 1× PBS.
5. Remove the PBS, and block the cells by adding 100 myl of to each well. Incubate for 30 minutes at RT.
6. Remove the blocking buffer and add 50 myl of the optimally titrated primary antibody (diluted in Stain Buffer) to each well, and incubate for 1 hour at RT.
7. Remove the primary antibody, and wash the wells three times with 100 myl of 1× PBS.
8. Remove the PBS, and add the second step reagent at its optimally titrated concentration in 50 myl to each well, and incubate in the dark for 1 hour at RT.
9. Remove the second step reagent, and wash the wells three times with 100 myl of 1× PBS.
10. Remove the PBS, and counter-stain the nuclei by adding 200 myl per well of 2 myg/ml Hoechst 33342 in 1× PBS to each well at least 15 minutes before imaging.
11. View and analyze the cells on an appropriate imaging instrument.
Comment

Related Products: ABIN968537, ABIN967389

Restrictions For Research Use only
Format Liquid
Concentration 250 µg/ml
Buffer Aqueous buffered solution containing BSA, glycerol.
Preservative Sodium azide
Precaution of Use This product contains sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
Storage -20 °C
Supplier Images
anti-Nuclear Factor-KB P65 (NFkBP65) (AA 136-224) antibody Immunofluorescent staining of A549 (ATCC CCL-185) cells. Cells were seeded in a 96 well imaging plate at ~ 10 000 cells per well. After overnight incubation, cells were either mock treated (media, left) or exposed to TNF (20ng/ml, right) for 15 minutes. After treatment cells were stained using the Triton™ X-100 perm protocol and the anti-NF-kappaB antibody. The second step reagent was Alexa-Fluor® 488 goat anti-mouse IgG (Invitrogen). The image was taken on a BD Pathway™ 855 Bioimager with a 20x objective. This antibody also stains U-2 OS (ATCC HTB-96) and HeLa (ATCC CCL-2) cells and can be used with either perm protocol.
anti-Nuclear Factor-KB P65 (NFkBP65) (AA 136-224) antibody (2) Western blot analysis of NF-kappaB p65 on a Jurkat lysate. Lane 1: 1:250, lane 2: 1:500, lane 3: 1:1000 dilution of NF-kappaB p65 antibody.
anti-Nuclear Factor-KB P65 (NFkBP65) (AA 136-224) antibody (3) anti-Nuclear Factor-KB P65 (NFkBP65) (AA 136-224) antibody (Image 3)
Product cited in: Kieran, Blank, Logeat et al.: "The DNA binding subunit of NF-kappa B is identical to factor KBF1 and homologous to the rel oncogene product." in: Cell, Vol. 62, Issue 5, pp. 1007-18, 1990 (PubMed).

Shirakawa, Mizel: "In vitro activation and nuclear translocation of NF-kappa B catalyzed by cyclic AMP-dependent protein kinase and protein kinase C." in: Molecular and cellular biology, Vol. 9, Issue 6, pp. 2424-30, 1989 (PubMed).

Baeuerle, Baltimore: "Activation of DNA-binding activity in an apparently cytoplasmic precursor of the NF-kappa B transcription factor." in: Cell, Vol. 53, Issue 2, pp. 211-7, 1988 (PubMed).

Pimentel-Muiños, Seed: "Regulated commitment of TNF receptor signaling: a molecular switch for death or activation." in: Immunity, Vol. 11, Issue 6, pp. 783-93, 2000 (PubMed).

Nishibe, Parry, Ishida et al.: "Oncostatin M promotes biphasic tissue factor expression in smooth muscle cells: evidence for Erk-1/2 activation." in: Blood, Vol. 97, Issue 3, pp. 692-9, 2001 (PubMed).

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