Recombinant Anti-NFkB p100/NFKB2 antibody [EPR4686] (ab109440)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR4686] to NFkB p100/NFKB2
- Suitable for: ICC/IF, WB
- Knockout validated
- Reacts with: Human
Overview
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Product name
Anti-NFkB p100/NFKB2 antibody [EPR4686]
See all NFkB p100/NFKB2 primary antibodies -
Description
Rabbit monoclonal [EPR4686] to NFkB p100/NFKB2 -
Host species
Rabbit -
Tested applications
Suitable for: ICC/IF, WBmore details
Unsuitable for: Flow Cyt or IHC-P -
Species reactivity
Reacts with: Human
Predicted to work with: Mouse -
Immunogen
Synthetic peptide within Human NFkB p100/NFKB2 aa 700 to the C-terminus. The exact sequence is proprietary.
Database link: Q00653 -
Positive control
- WB: Jurkat, HeLa, ECV-304, HepG2, HCT116 and MCF7 cell lysates ICC/IF: Wild-type HAP1 cells.
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General notes
Rat: We have preliminary internal testing data to indicate this antibody may not react with this species. Please contact us for more information.
This product was previously labelled as NFkB p100
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.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at -20°C. Stable for 12 months at -20°C. -
Storage buffer
pH: 7.20
Preservative: 0.05% Sodium azide
Constituents: 0.1% BSA, 40% Glycerol (glycerin, glycerine), 9.85% Tris glycine, 50% Tissue culture supernatant -
Concentration information loading...
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Purity
Tissue culture supernatant -
Clonality
Monoclonal -
Clone number
EPR4686 -
Isotype
IgG -
Research areas
Associated products
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Compatible Secondaries
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Isotype control
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KO cell lines
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KO cell lysates
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Positive Controls
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab109440 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 |
1/250.
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WB | (1) |
1/10000 - 1/50000. Detects a band of approximately 110 kDa (predicted molecular weight: 97 kDa).
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Notes |
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ICC/IF
1/250. |
WB
1/10000 - 1/50000. Detects a band of approximately 110 kDa (predicted molecular weight: 97 kDa). |
Target
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Relevance
NF-kappa-B is a pleiotropic transcription factor present in almost all cell types and is the endpoint of a series of signal transduction events that are initiated by a vast array of stimuli related to many biological processes 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. 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. In a non-canonical activation pathway, the MAP3K14-activated CHUK/IKKA homodimer phosphorylates NFKB2/p100 associated with RelB, inducing its proteolytic processing to NFKB2/p52 and the formation of NF-kappa-B RelB-p52 complexes. The NF-kappa-B heterodimeric RelB-p52 complex is a transcriptional activator. The NF-kappa-B p52-p52 homodimer is a transcriptional repressor. NFKB2 appears to have dual functions such as cytoplasmic retention of attached NF-kappa-B proteins by p100 and generation of p52 by a cotranslational processing. The proteasome-mediated process ensures the production of both p52 and p100 and preserves their independent function. p52 binds to the kappa-B consensus sequence 5'-GGRNNYYCC-3', located in the enhancer region of genes involved in immune response and acute phase reactions. p52 and p100 are respectively the minor and major form; the processing of p100 being relatively poor. Isoform p49 is a subunit of the NF-kappa-B protein complex, which stimulates the HIV enhancer in synergy with p65. In concert with RELB, regulates the circadian clock by repressing the transcriptional activator activity of the CLOCK-ARNTL/BMAL1 heterodimer. -
Cellular localization
Cytoplasmic and Nuclear -
Database links
- Entrez Gene: 4791 Human
- Entrez Gene: 18034 Mouse
- Omim: 164012 Human
- SwissProt: Q00653 Human
- SwissProt: Q9WTK5 Mouse
- Unigene: 73090 Human
- Unigene: 102365 Mouse
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Alternative names
- CVID10 antibody
- DNA binding factor KBF2 antibody
- H2TF1 antibody
see all
Images
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Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: NFκB p100 knockout HAP1 cell lysate (20 µg)
Lane 3: Jurkat cell lysate (20 µg)
Lane 4: HeLa cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab109440 observed at 100 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab109440 was shown to specifically react with NFκB p100 when NFκB p100 knockout samples were used. Wild-type and NFκB p100 knockout samples were subjected to SDS-PAGE. ab109440 and ab109440 (loading control to GAPDH) were diluted 1/10000 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 1/10000 dilution for 1 h at room temperature before imaging. -
ab109440 staining NFkB p100/p52 in wild-type HAP1 cells (top panel) and NFkB p100/p52 knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde (10min), 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 with ab109440 at 1/250 dilution and ab195889 at 1/250 dilution (shown in pseudo colour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.
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All lanes : Anti-NFkB p100/NFKB2 antibody [EPR4686] (ab109440) at 1/1000 dilution
Lane 1 : Wild-type HCT116 cell lysate
Lane 2 : NFKB2 CRISPR/Cas9 edited HCT116 cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 97 kDa
Observed band size: 120 kDa why is the actual band size different from the predicted?Lanes 1- 2: Merged signal (red and green). Green - ab109440 observed at 120 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.
ab109440 was shown to react with NFkB p100/NFKB2 in wild-type HCT116 cells in western blot. The band observed in CRISPR/Cas9 edited cell line ab266883 (CRISPR/Cas9 edited cell lysate ab257245) lane below 97kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HCT116 and NFKB2 CRISPR/Cas9 edited HCT116 cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab109440 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. 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 in 20000 dilution for 1 hour at room temperature before imaging.
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All lanes : Anti-NFkB p100/NFKB2 antibody [EPR4686] (ab109440) at 1/1000 dilution
Lane 1 : Wild-type HepG2 cell lysate
Lane 2 : NFKB2 CRISPR/Cas9 edited HepG2 cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 97 kDa
Observed band size: 120 kDa why is the actual band size different from the predicted?Lanes 1- 2: Merged signal (red and green). Green - ab109440 observed at 120 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.
ab109440 was shown to react with NFkB p100/NFKB2 in wild-type HepG2 cells in western blot. The band observed in CRISPR/Cas9 edited cell line ab262323 (CRISPR/Cas9 edited cell lysate ab257247) lane below 97kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HepG2 and NFKB2 CRISPR/Cas9 edited HepG2 cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab109440 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. 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 in 20000 dilution for 1 hour at room temperature before imaging.
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All lanes : Anti-NFkB p100/NFKB2 antibody [EPR4686] (ab109440) at 1/10000 dilution
Lane 1 : Jurkat cell lysate
Lane 2 : HeLa cell lysate
Lane 3 : ECV-304 cell lysate
Lane 4 : MCF7 cell lysate
Lysates/proteins at 10 µg per lane.
Predicted band size: 97 kDa
Observed band size: 110 kDa why is the actual band size different from the predicted?
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (2)
ab109440 has been referenced in 2 publications.
- Elzaiat M et al. High-throughput Exploration of the Network Dependent on AKT1 in Mouse Ovarian Granulosa Cells. Mol Cell Proteomics 18:1307-1319 (2019). PubMed: 30992313
- Sikorski K et al. A high-throughput pipeline for validation of antibodies. Nat Methods 15:909-912 (2018). PubMed: 30377371