Anti-NFkB p105 / p50 antibody - ChIP Grade (ab7971)
- Product nameAnti-NFkB p105 / p50 antibody - ChIP GradeSee all NFkB p105 / p50 primary antibodies ...
- DescriptionRabbit polyclonal to NFkB p105 / p50 - ChIP Grade
- SpecificityReacts with NFkB p50 and p105
- Tested applicationsIHC-Fr, IP, WB, ChIP, Flow Cyt, EMSA, IHC-P, ICC/IF more details
- Species reactivityReacts with: Mouse, Rat, Human
Synthetic peptide derived from the region aa330 to aa433 of the Human NFkB p50 subunit.
- Positive control
- K-562, A-431, HeLa
- General notes
Members of the rel/NFkappaB family of transcription factors are involved in the regulation of cellular responses, such as growth, development, and the inflammatory response. They share a structural motif known as the rel homology region (RHR), the C-terminal one third of which mediates protein dimerization. Complexes of p50 (NF-kB1) or p52 (NF-kB2) are generated through the processing of p105 and p100 precursors, respectively. These are usually associated with members of the Rel family (p65, c-Rel, Rel B). The homo- and heterodimer formed through combinations of NF-kB/Rel proteins bind distinct kB sites to regulate the transcription of different genes.
- Storage instructionsStore at +4°C short term (1-2 weeks). Store at -20°C or -80°C. Avoid freeze / thaw cycle.
- Storage bufferPreservative: 0.1% Sodium azide
Constituents: PBS, 0.2% Gelatin
- Concentration information loading...
- PurityIgG fraction
- Clonality Polyclonal
Our Abpromise guarantee covers the use of ab7971 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC-Fr||IHC-Fr: Use at an assay dependent dilution.|
|IP||IP: Use at an assay dependent dilution.|
|WB||WB: 1/400. Predicted molecular weight: 50 kDa.Can be blocked with NFkB p105 / p50 peptide (ab8006). PubMed: 16395278|
|ChIP||ChIP: Use at an assay dependent dilution.|
|Flow Cyt||Flow Cyt: Use at an assay dependent concentration.|
|EMSA||EMSA: Use at an assay dependent concentration. PubMed: 22553204|
|ICC/IF||ICC/IF: Use at an assay dependent concentration.|
- FunctionNF-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 RelB-p50 complexes are transcriptional activators. The NF-kappa-B p50-p50 homodimer is a transcriptional repressor, but can act as a transcriptional activator when associated with BCL3. NFKB1 appears to have dual functions such as cytoplasmic retention of attached NF-kappa-B proteins by p105 and generation of p50 by a cotranslational processing. The proteasome-mediated process ensures the production of both p50 and p105 and preserves their independent function, although processing of NFKB1/p105 also appears to occur post-translationally. p50 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. In a complex with MAP3K8, NFKB1/p105 represses MAP3K8-induced MAPK signaling; active MAP3K8 is released by proteasome-dependent degradation of NFKB1/p105.
- Sequence similaritiesContains 7 ANK repeats.
Contains 1 death domain.
Contains 1 RHD (Rel-like) domain.
- DomainThe C-terminus of p105 might be involved in cytoplasmic retention, inhibition of DNA-binding, and transcription activation.
Glycine-rich region (GRR) appears to be a critical element in the generation of p50.
modificationsWhile translation occurs, the particular unfolded structure after the GRR repeat promotes the generation of p50 making it an acceptable substrate for the proteasome. This process is known as cotranslational processing. The processed form is active and the unprocessed form acts as an inhibitor (I kappa B-like), being able to form cytosolic complexes with NF-kappa B, trapping it in the cytoplasm. Complete folding of the region downstream of the GRR repeat precludes processing.
Phosphorylation at 'Ser-903' and 'Ser-907' primes p105 for proteolytic processing in response to TNF-alpha stimulation. Phosphorylation at 'Ser-927' and 'Ser-932' are required for BTRC/BTRCP-mediated proteolysis.
Polyubiquitination seems to allow p105 processing.
S-nitrosylation of Cys-61 affects DNA binding.
- Cellular localizationNucleus. Cytoplasm. Nuclear, but also found in the cytoplasm in an inactive form complexed to an inhibitor.
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Anti-NFkB p105 / p50 antibody - ChIP Grade images
HeLa cells were fixed with 4% formaldehyde in PEM buffer. The coverslip was incubated in blocking buffer of 5% powdered milk in TBS-T plus 0.02% sodium azide for 1 hour at room temperature. Blocking buffer was removed and primary antibody was added at a dilution of 1/500 and incubated overnight at 4 degrees celsius. The coverslips were then washed 4-5 times with blocking buffer for 5 minutes. Secondary antibody, goat anti-rabbit Alexa 594, was added at a dilution of 1/1000 and incubated at room temperature for one hour. From this point on coverslips were covered with foil to protect them from light. They were washed 5 times with TBS-T and then one time with PEM, for 5 minutes each wash. The coverslips were fixed 10-30 minutes in 4% formaldehyde in PEM buffer, then washed 3 times with PEM buffer for 5 minutes. 0.1M ammonium chloride in PEM buffer was added for 10 minutes to quench auto-florescence, and then slips were washed 2 times for 5 minutes in PEM followed by 3 washes for 5 minutes in TBS-T. Coverslips were then counterstained with DAPI in TBS-T for 1-2 minutes, TBS-T was then added and the coverslips mounted. Red indicates staining by ab7971, blue staining by DAPI.
X-ChIP with ab7971 showing enrichment of NFkB p105 / p50 at regions upstream of transcription start site of the NFKBIA and IL8 genes. Chromatin was extracted from monocytes treated with LPS and the proteins were cross-linked to DNA with formaldehyde for 15 minutes. The immunoprecipitation step involved incubation of the cross-linked chromatin with ab7971 for 16 hours at 4°C. Please see abreview by Genpathway for additional details.
ICC/IF image of ab7971 stained HepG2 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab7971, 5µg/ml) overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti-rabbit IgG - H&L, pre-adsorbed (ab96899) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
ab7971 staining NFkB p105 / p50 in Human Jurkat cells by Flow Cytometry. Cells were prepared in a phosphate buffered solution containing 0.1% sodium azide with FBS, fixed with paraformaldehyde and permeabilized with Triton X-100 and NP40. The sample was incubated with the primary antibody (1/100 in wash buffer) for 24 hours at 4°C. A FITC-conjugated Goat anti-rabbit Ig (1/100) was used as the secondary antibody.
Gating Strategy: Isolate cell population from plot of SSC-A / FSA-A
References for Anti-NFkB p105 / p50 antibody - ChIP Grade (ab7971)
This product has been referenced in:
- Tang JR et al. The NF-?B inhibitory proteins I?Ba and I?Bß mediate disparate responses to inflammation in fetal pulmonary endothelial cells. J Immunol 190:2913-23 (2013). Rat . Read more (PubMed: 23418625) »
- Lakota K et al. Serum amyloid A activation of human coronary artery endothelial cells exhibits a neutrophil promoting molecular profile. Microvasc Res N/A:N/A (2013). Read more (PubMed: 23938271) »