Anti-NFkB p105 / p50 antibody - ChIP Grade (ab7971)

Overview

  • Product nameAnti-NFkB p105 / p50 antibody - ChIP GradeSee all NFkB p105 / p50 primary antibodies ...
  • Description
    Rabbit 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/IFmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide derived from the region aa330 to aa433 of the Human NFkB p50 subunit.

  • Positive control
    • K-562, A-431, HeLa

Properties

Applications

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.

Application Abreviews Notes
IHC-Fr Use at an assay dependent dilution.
IP Use at an assay dependent dilution.
WB 1/400. Predicted molecular weight: 50 kDa.Can be blocked with NFkB p105 / p50 peptide (ab8006). PubMed: 16395278
ChIP Use at an assay dependent dilution.
Flow Cyt Use at an assay dependent concentration.
EMSA Use at an assay dependent concentration. PubMed: 22553204
IHC-P 1/100.
ICC/IF Use at an assay dependent concentration.

Target

  • 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.
  • Post-translational
    modifications
    While 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.
  • Information by UniProt
  • Database links
  • Alternative names
    • DKFZp686C01211 antibody
    • DNA binding factor KBF1 antibody
    • DNA binding factor KBF1 EBP1 antibody
    • DNA binding factor KBF1 EBP1 antibody
    • DNA-binding factor KBF1 antibody
    • EBP 1 antibody
    • EBP-1 antibody
    • EBP1 antibody
    • KBF1 antibody
    • MGC54151 antibody
    • NF kappa B antibody
    • NF kappabeta antibody
    • NF kB1 antibody
    • NFKB 1 antibody
    • NFKB p105 antibody
    • NFKB p50 antibody
    • NFKB1 antibody
    • NFKB1_HUMAN antibody
    • Nuclear factor kappa B DNA binding subunit antibody
    • Nuclear factor NF kappa B p105 subunit antibody
    • Nuclear factor NF kappa B p50 subunit antibody
    • Nuclear factor NF-kappa-B p50 subunit antibody
    • Nuclear factor of kappa light polypeptide gene enhancer in B cells 1 antibody
    • Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 antibody
    • p84/NF-kappa-B1 p98 antibody
    see all

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.

    See Abreview

  • 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:
  • Luo X  et al. Dynamic reorganization of the AC16 cardiomyocyte transcriptome in response to TNFa signaling revealed by integrated genomic analyses. BMC Genomics 15:155 (2014). WB . Read more (PubMed: 24564208) »
  • Tzeng HP  et al. Dysferlin mediates the cytoprotective effects of TRAF2 following myocardial ischemia reperfusion injury. J Am Heart Assoc 3:e000662 (2014). ChIP ; Mouse . Read more (PubMed: 24572254) »

See all 31 Publications for this product

Product Wall


EMSA is not an application that we test our antibodies in, in house. All information that we have for our products for EMSA come from publications. It is therefore a shame that the publication for this product does not have more information.
...

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Application ChIP
Detection step Real-time PCR
Sample Mouse Cell lysate - whole cell (Colon)
Specification Colon
Type Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
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Submitted May 28 2014

ChIP

Excellent
Abreviews
Abcam guarantees this product to work in the species/application used in this Abreview.
Application ChIP
Detection step Real-time PCR
Sample Mouse Cell lysate - whole cell (mouse microglia cell line)
Specification mouse microglia cell line
Negative control IgG
Type Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: 1% formaldehyde
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Submitted Jun 07 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Human Cell lysate - whole cell (PBMC)
Loading amount 30 µg
Specification PBMC
Gel Running Conditions Reduced Denaturing (10)
Blocking step Odyssey Blocking Buffer as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 25°C
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Submitted May 14 2013

Thank you for contacting us.

There are 3 antibodies we can offer ab31410, ab28849 and ab7971. The immunogen of these antibodies is significantly similar to Porcine protein http://www.uniprot.org/uniprot/Q0PHA8 so these antibodies might cross r...

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application ChIP
Sample Human Cell lysate - whole cell (Breast cell line)
Specification Breast cell line
Type Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Detection step Real-time PCR
Username

Mr. Ohad Tarcic

Verified customer

Submitted Jun 08 2012

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunocytochemistry/ Immunofluorescence
Sample Mouse Cell (Pancreas)
Specification Pancreas
Fixative Methanol
Permeabilization No
Blocking step Serum as blocking agent for 30 minute(s) · Concentration: 10%
Username

Mr. Ohad Tarcic

Verified customer

Submitted Jun 08 2012

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Human Cell lysate - whole cell (breast cell line)
Loading amount 100000 cells
Specification breast cell line
Gel Running Conditions Reduced Denaturing
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3%
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Verified customer

Submitted May 11 2012

Thank you for contacting us. Because we carry over 70,000 products, it isn't feasible for us to keep small sample sizes of our products.

We are happy to reassure our customers that all of our products are covered by our Abpromise, which guar...

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunoprecipitation
Sample Human Cell lysate - whole cell (Breast cell line)
Total protein in input 100000 cells
Specification Breast cell line
Immuno-precipitation step Protein A
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Verified customer

Submitted Apr 27 2012

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"