Recombinant Anti-NF-kB p65 antibody [E379] - BSA and Azide free (ab207297)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [E379] to NF-kB p65 - BSA and Azide free
- Suitable for: ICC/IF, IP, WB, IHC-P
- Knockout validated
- Reacts with: Mouse, Human
Related conjugates and formulations
Overview
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Product name
Anti-NF-kB p65 antibody [E379] - BSA and Azide free
See all NF-kB p65 primary antibodies -
Description
Rabbit monoclonal [E379] to NF-kB p65 - BSA and Azide free -
Host species
Rabbit -
Tested applications
Suitable for: ICC/IF, IP, WB, IHC-Pmore details
Unsuitable for: Flow Cyt -
Species reactivity
Reacts with: Mouse, Human -
Immunogen
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- WB: Wild-type HAP1 cell lysate. HeLa, MEF, RAW 264.7 and A431 cell lysate; Human fetal brain, kidney and lung tissue lysates. IHC-P: Human breast carcinoma and colon carcinoma tissue. Mouse colon tissue. Mouse spleen, human tonsil and human prostatic hyperplasia tissues. ICC/IF: HeLa and NIH/3T3 cells. IP: NF-kB p65 IP in HeLa whole cell lysate (ab150035).
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General notes
ab207297 is the carrier-free version of ab32536.
Our carrier-free antibodies are typically supplied in a PBS-only formulation, purified and free of BSA, sodium azide and glycerol. The carrier-free buffer and high concentration allow for increased conjugation efficiency.
This conjugation-ready format is designed for use with fluorochromes, metal isotopes, oligonucleotides, and enzymes, which makes them ideal for antibody labelling, functional and cell-based assays, flow-based assays (e.g. mass cytometry) and Multiplex Imaging applications.
Use our conjugation kits for antibody conjugates that are ready-to-use in as little as 20 minutes with <1 minute hands-on-time and 100% antibody recovery: available for fluorescent dyes, HRP, biotin and gold.
This product is compatible with the Maxpar® Antibody Labeling Kit from Fluidigm, without the need for antibody preparation. Maxpar® is a trademark of Fluidigm Canada Inc.
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
Rat: We have preliminary internal testing data to indicate this antibody may not react with this species. Please contact us for more information.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C. Do Not Freeze. -
Storage buffer
pH: 7.20
Constituent: PBS -
Carrier free
Yes -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
E379 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
- Alexa Fluor® 488 Anti-NF-kB p65 antibody [E379] (ab190205)
- Alexa Fluor® 647 Anti-NF-kB p65 antibody [E379] (ab190589)
- Biotin Anti-NF-kB p65 antibody [E379] (ab205823)
- PE Anti-NF-kB p65 antibody [E379] (ab208750)
- Alexa Fluor® 594 Anti-NF-kB p65 antibody [E379] (ab214846)
- Anti-NF-kB p65 antibody [E379] (ab32536)
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Compatible Secondaries
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Conjugation kits
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Isotype control
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Positive Controls
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Recombinant Protein
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab207297 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Application | Abreviews | Notes |
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ICC/IF |
Use at an assay dependent concentration.
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IP |
Use at an assay dependent concentration.
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WB |
Use at an assay dependent concentration. Detects a band of approximately 65 kDa (predicted molecular weight: 65 kDa).
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IHC-P |
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
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Notes |
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ICC/IF
Use at an assay dependent concentration. |
IP
Use at an assay dependent concentration. |
WB
Use at an assay dependent concentration. Detects a band of approximately 65 kDa (predicted molecular weight: 65 kDa). |
IHC-P
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol. |
Target
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Function
NF-kappa-B is a pleiotropic transcription factor which is present in almost all cell types and is involved in many biological processed such as inflammation, immunity, differentiation, cell growth, tumorigenesis and apoptosis. NF-kappa-B is a homo- or heterodimeric complex formed by the Rel-like domain-containing proteins RELA/p65, RELB, NFKB1/p105, NFKB1/p50, REL and NFKB2/p52 and the heterodimeric p65-p50 complex appears to be most abundant one. The dimers bind at kappa-B sites in the DNA of their target genes and the individual dimers have distinct preferences for different kappa-B sites that they can bind with distinguishable affinity and specificity. Different dimer combinations act as transcriptional activators or repressors, respectively. NF-kappa-B is controlled by various mechanisms of post-translational modification and subcellular compartmentalization as well as by interactions with other cofactors or corepressors. NF-kappa-B complexes are held in the cytoplasm in an inactive state complexed with members of the NF-kappa-B inhibitor (I-kappa-B) family. In a conventional activation pathway, I-kappa-B is phosphorylated by I-kappa-B kinases (IKKs) in response to different activators, subsequently degraded thus liberating the active NF-kappa-B complex which translocates to the nucleus. NF-kappa-B heterodimeric p65-p50 and p65-c-Rel complexes are transcriptional activators. The NF-kappa-B p65-p65 complex appears to be involved in invasin-mediated activation of IL-8 expression. The inhibitory effect of I-kappa-B upon NF-kappa-B the cytoplasm is exerted primarily through the interaction with p65. p65 shows a weak DNA-binding site which could contribute directly to DNA binding in the NF-kappa-B complex. Associates with chromatin at the NF-kappa-B promoter region via association with DDX1. -
Sequence similarities
Contains 1 RHD (Rel-like) domain. -
Domain
the 9aaTAD motif is a transactivation domain present in a large number of yeast and animal transcription factors. -
Post-translational
modificationsUbiquitinated, leading to its proteasomal degradation. Degradation is required for termination of NF-kappa-B response.
Monomethylated at Lys-310 by SETD6. Monomethylation at Lys-310 is recognized by the ANK repeats of EHMT1 and promotes the formation of repressed chromatin at target genes, leading to down-regulation of NF-kappa-B transcription factor activity. Phosphorylation at Ser-311 disrupts the interaction with EHMT1 without preventing monomethylation at Lys-310 and relieves the repression of target genes.
Phosphorylation at Ser-311 disrupts the interaction with EHMT1 and promotes transcription factor activity (By similarity). Phosphorylation on Ser-536 stimulates acetylation on Lys-310 and interaction with CBP; the phosphorylated and acetylated forms show enhanced transcriptional activity.
Reversibly acetylated; the acetylation seems to be mediated by CBP, the deacetylation by HDAC3. Acetylation at Lys-122 enhances DNA binding and impairs association with NFKBIA. Acetylation at Lys-310 is required for full transcriptional activity in the absence of effects on DNA binding and NFKBIA association. Acetylation can also lower DNA-binding and results in nuclear export. Interaction with BRMS1 promotes deacetylation of 'Lys-310'. -
Cellular localization
Nucleus. Cytoplasm. Nuclear, but also found in the cytoplasm in an inactive form complexed to an inhibitor (I-kappa-B). Colocalized with RELA in the nucleus upon TNF-alpha induction. - Information by UniProt
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Database links
- Entrez Gene: 5970 Human
- Entrez Gene: 19697 Mouse
- Omim: 164014 Human
- SwissProt: Q04206 Human
- SwissProt: Q04207 Mouse
- Unigene: 502875 Human
- Unigene: 249966 Mouse
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Alternative names
- Avian reticuloendotheliosis viral (v rel) oncogene homolog A antibody
- MGC131774 antibody
- NF kappa B p65delta3 antibody
see all
Images
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This WB data was generated using the same anti-NF-kB p65 antibody clone, E379, in a different buffer formulation (cat# ab32536).
Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: NF-kB p65 knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: A431 cell lysate (20 µg)Lanes 1 - 4: Merged signal (red and green). Green - ab32536 observed at 70 kDa. Red - ab8245 loading control, observed at 37 kDa.
ab32536 was shown to specifically react with NF-kB p65 in wild-type HAP1 cells. No band was observed when NF-kB p65 knockout samples were used. Wild-type and NF-kB p65 knockout samples were subjected to SDS-PAGE. ab32536 (NF-kB p65) and ab8245 (loading control to GAPDH) were diluted to 1/50 000 and 1/2000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging. -
Immunocytochemistry/ Immunofluorescence - Anti-NF-kB p65 antibody [E379] - BSA and Azide free (ab207297)
Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling NF-kB p65 with purified ab32536 at 1:100 dilution. Cells were fixed in 100% Methanol. ab150077 Goat anti rabbit IgG(Alexa Fluor® 488) was used as the secondary antibody at 1:1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32536).
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NF-kB p65 antibody [E379] - BSA and Azide free (ab207297)
This data was generated using the same anti-NF-kB p65 antibody clone, E379, in a different buffer formulation (ab32536).
Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling NF-kB p65 with ab32536 at 1/5000 (0.098 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Cytoplasmic staining on mouse spleen. The section was incubated with ab32536 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).
Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins
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Immunocytochemistry/ Immunofluorescence - Anti-NF-kB p65 antibody [E379] - BSA and Azide free (ab207297)
This data was generated using the same anti-NF-kB p65 antibody clone, E379, in a different buffer formulation (ab32536).
Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 cells labelling NF-kB p65 with ab32536 at 1/100 (4.89 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green). Confocal image showing the signal translocated from the cytoplasm into the nucleus in NIH/3T3 cells after the treatment with TNF-alpha (50 ng/ml) for 20 min. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).
Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution in treated (right) and untreated cells (left).
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ab32536 (purified) at 1:30 dilution (2μg) immunoprecipitating NF-kB p65 in HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate.
Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10μg
Lane 2 (+): ab32536 & HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32536 in HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST.This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32536).
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NF-kB p65 antibody [E379] - BSA and Azide free (ab207297)
This data was generated using the same anti-NF-kB p65 antibody clone, E379, in a different buffer formulation (ab32536).
Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling NF-kB p65 with ab32536 at 1/5000 (0.098 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Cytoplasmic staining on human tonsil. The section was incubated with ab32536 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).
Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NF-kB p65 antibody [E379] - BSA and Azide free (ab207297)
This data was generated using the same anti-NF-kB p65 antibody clone, E379, in a different buffer formulation (ab32536).
Immunohistochemical analysis of paraffin-embedded Human prostatic hyperplasia tissue labeling NF-kB p65 with ab32536 at 1/5000 (0.098 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Cytoplasmic staining on human prostatic hyperplasia. The section was incubated with ab32536 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).
Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NF-kB p65 antibody [E379] - BSA and Azide free (ab207297)
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human colon carcinoma tissue sections labeling NF-kB p65 with Purified ab32536 at 1:2000 dilution (0.2 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32536).
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Immunocytochemistry/ Immunofluorescence - Anti-NF-kB p65 antibody [E379] - BSA and Azide free (ab207297)
Clone E379 (ab207297) has been successfully conjugated by Abcam. This image was generated using Anti-NF-kB p65 antibody [E379] (Alexa Fluor® 647). Please refer to ab190589 for protocol details.
ab190589 staining NF-kB p65 in HeLa cells. The cells were fixed with 4% formaldehyde (10 min), permeabilized in 0.1% Triton X-100 for 5 minutes and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab190589 at a working dilution of 1 in 100 (shown in red) and ab195887, Mouse monoclonal [DM1A] to alpha Tubulin (Alexa Fluor® 488, shown in green) at 2µg/ml overnight at +4°C. Nuclear DNA was labelled in blue with DAPI.
This product also gave a positive signal in 100% methanol (5 min) fixed HeLa cells under the same testing conditions.
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
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Immunocytochemistry/ Immunofluorescence - Anti-NF-kB p65 antibody [E379] - BSA and Azide free (ab207297)
Clone E379 (ab207297) has been successfully conjugated by Abcam. This image was generated using Anti-NF-kB p65 antibody [E379] (PE). Please refer to ab208750 for protocol details.
ab208750 staining NF-kB p65 in HeLa cells. The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab208750 at 1/500 dilution (Pseudocolored in green) and ab195884, Rat monoclonal to Tubulin (Alexa Fluor® 647), at 1/250 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
This product also gave a positive signal under the same testing conditions in HeLa cells fixed with 100% methanol (5min).
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Immunocytochemistry/ Immunofluorescence - Anti-NF-kB p65 antibody [E379] - BSA and Azide free (ab207297)
Clone E379 (ab207297) has been successfully conjugated by Abcam. This image was generated using Anti-NF-kB p65 antibody [E379] (Alexa Fluor® 488). Please refer to ab190205 for protocol details.
ab190205 staining NF-kB p65 in HeLa cells. The cells were fixed with 100% methanol (5 min), permeabilized in 0.1% Triton X-100 for 5 minutes and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with ab190205 at a working dilution of 1 in 50 (shown in green) and ab195889, Mouse monoclonal [DM1A] to alpha Tubulin (Alexa Fluor® 594, shown in red) at a dilution of 1 in 250 overnight at +4°C. Nuclear DNA was labelled in blue with DAPI.
This product also gave a positive signal in 4% formaldehyde (10 min) fixed HeLa cells under the same testing conditions.
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
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Immunocytochemistry/ Immunofluorescence - Anti-NF-kB p65 antibody [E379] - BSA and Azide free (ab207297)Image from Ali Ahmed Atef Ahmed et al. PLoS ONE 11.4 (2016): e0154278. Fig 8. doi: 10.1371/journal.pone.0154278.
Immunocytochemistry/ Immunofluorescence analysis of human cancer cells labeling NF-kB p65 with unpurified ab32536. Briefly, the tested cells were seeded on coverslips treated with HCl and ethanol, and autoclaved prior to use. Immunostaining of the p65 subunit of NF-κB was done by permeabilizing the cells with Triton X-10, then by treating the cells with anti-NF-κB p65 rabbit monoclonal primary antibody [E379] (ab32536), followed by Alexa Fluor® 488 Donkey anti-rabbit IgG secondary antibody. Nuclei of cells were stained with DAPI. Images were acquired using fluorescence microscope.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32536).
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NF-kB p65 antibody [E379] - BSA and Azide free (ab207297)
Immunohistochemical analysis of paraffin-embedded human Breast carcinoma using unpurified anti-NF-kB p65 Rabbit Monoclonal Antibody
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32536).
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NF-kB p65 antibody [E379] - BSA and Azide free (ab207297)
This IHC data was generated using the same anti-NF-kB p65 antibody clone, E379, in a different buffer formulation (cat# ab32536).
Immunohistochemical analysis of colon sections from mice, staining NF-kB p65 with ab32536.
Antigen retrieval was performed by microwave heating in citrate buffer, pH 6. Sections were incubated overnight with primary antibody (1/250) and staining was detected using ab80437 EXPOSE Rabbit specific HRP/DAB detection IHC kit.
Protocols
Datasheets and documents
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Datasheet download
Certificate of Compliance
References (61)
ab207297 has been referenced in 61 publications.
- Chen X et al. KLF4 downregulates FGF21 to activate inflammatory injury and oxidative stress of LPS-induced ATDC5 cells via SIRT1/NF-?B/p53 signaling. Mol Med Rep 25:N/A (2022). WB ; Human . PubMed: 35293599
- Hu Y et al. Curcumol enhances cisplatin sensitivity of gastric cancer: involvement of microRNA-7 and the nuclear factor-kappa B/snail family transcriptional repressor 1 axis. Bioengineered 13:11668-11683 (2022). WB ; Human . PubMed: 35510522
- Xie Z et al. Calcium dobesilate alleviates renal dysfunction and inflammation by targeting nuclear factor kappa B (NF-κB) signaling in sepsis-associated acute kidney injury. Bioengineered 13:2816-2826 (2022). PubMed: 35038964
- Wang X et al. Study on the intervention effect and mechanism of bacillus Calmette-Guerin polysaccharide and nucleic acid injection on atopic dermatitis by targeting the transient receptor potential vanilloid subtype 1 pathway. Ann Transl Med 10:608 (2022). PubMed: 35722408
- Zou T et al. Salvianolic acid B inhibits RAW264.7 cell polarization towards the M1 phenotype by inhibiting NF-κB and Akt/mTOR pathway activation. Sci Rep 12:13857 (2022). PubMed: 35974091