Ordering Information
- : MAB3026
- : 100 µg
- : 1 mg/mL
Product Images
Western Blot Analysis:
PC12 lysate was resolved by electrophoresis, transferred to PVDF membrane and probed with anti-NF KAPPA B, P65 (1:500 dilu...
Western Blot Analysis:
Representative image from a previous lot.
Huvec lysate (Lane 1), L6 lysate (Lane 2), PC12 lysate (Lane 3) were resolv...
Anti-NFκB, p65 subunit, active subunit, clone 12H11
| Species Reactivity | Key Applications | Host | Format | Antibody Type |
|---|---|---|---|---|
| H, M, R, Rb | EMSA, FC, IC, IF, IH, IH(P), WB | Mouse | Purified | Monoclonal Antibody |
Description:
Anti-NFκB, p65 subunit, active subunit, clone 12H11
Trade Name:
Chemicon (Millipore)
Specificity:
Recognizes an epitope overlapping the nuclear location signal (NLS) of the p65 subunit of the NFkB heterodimer. Thus it selectively binds to the activated form of NFkB (Zabel et al., 1993). The antibody can also be used for electrophoretic mobility supershift assays (EMSA). The nuclear factor kB (NFkB) is a sequence-specific DNA-binding protein (Sen & Baltimore, 1986). It is a pleiotropic transcription factor which is involved in the expression of a variety of cellular and viral genes (Lenardo & Baltimore, 1989). NFkB consists of two subunits which are named according to their molecular weight, p50 and p65 (Kawakami et al., 1988). The subunits are stabilized by a so-called inhibitory chain IkB (Baeuerle & Baltimore, 1988). In quiescent cells, NFkB resides in the cytosol in an inactive form which can be activated in vivo by treatment of cells with cytokines or protein kinase activators. In vitro, it is possible to generate the active form of NFkB by treatment with sodium deoxycholate, formadine or electrophoretic size fractionation. The active form of NFkB is a heterotetrameric protein, consisting of the two p50 and two p65 subunits (Lenardo et al., 1987). After activation NFkB translocates from the cytosol to the nucleus of the cell, binds to specific DNA sequences and initiates transcription.
Molecular Weight:
65 kDa
Epitope:
p65 subunit, active subunit
Immunogen:
Peptide corresponding to human p65 coupled to BSA.
Clone:
12H11
Isotype:
IgG3
Background Information:
The transcription factor NFkappaB (Nuclear Factor kappa B) is involved in the expression and regulation of a number of important cellular and physiological processes such as growth, development, apoptosis, immune and inflammatory response, and activation of various viral promoters including human immunodeficiency virus long terminal repeats. NFkappaB represents a group of structurally related and evolutionarily conserved proteins related to the proto-oncogene c-Rel with five members in mammals that include Rel (cRel), RelA (p65), RelB, NFkappaB1 (p50 and its precursor p105), and NFkappaB2 (p52 and its precursor p100). NFkappaB/Rel proteins exist as homo- or heterodimers to form transcriptionally competent or repressive complexes. Although most NFkappaB dimers are activators of transcription, the p50/50 and p52/52 homodimers can repress the transcription of their target genes. The p50/p65 heterodimer of NFkappaB is the most abundant in cells.
Species Reactivity:
- Human
- Mouse
- Rat
- Rabbit
Species Reactivity Note:
Human, rat, and rabbit. Expected to react with mouse based on sequence homology.
Application Notes:
Immunofluorescence:
A 1-10 μg/mL concentration of a previous lot was used in immunofluorescence.
Immunohistochemistry (paraffin sections):
A 5-10 μg/mL (APAAP) concentration of a previous lot was used in immunohistochemistry.
Immunohistochemistry (frozen sections):
A 5-10 μg/mL (APAAP) concentration of a previous lot was used in immunohistochemistry.
Immunohistochemistry:
The clone 12H11 works best in fresh frozen or acetone fixed human tissues, however some groups have had reactivity in traditional formalin fixed tissue when the tissue is of human origin. It is recommended that ABC or enhanced detection systems be employed for the best visualization in either acetone or formalin fixed tissues. The antibody reacts with human tissues best. Rat tissue will also react but at a lower affinity, and the antibody does not react with mouse, other species have not been examined. Microwave citric acid buffer or trypsin digestion antigen recovery have both been successful with 12H11 on formalin fixed tissues.
Western blot: 5-10 µg/mL (ECL)
Electrophoretic Mobility Supershift Assay:
A 0.5-1 μg/mL concentration of a previous lot was used in Supershift assay.
Flow cytometry:
A previous lot of this antibody was used in flow cytometry. Fixed cells only, acetone fixed cells.
Optimal working dilutions must be determined by end user.
A 1-10 μg/mL concentration of a previous lot was used in immunofluorescence.
Immunohistochemistry (paraffin sections):
A 5-10 μg/mL (APAAP) concentration of a previous lot was used in immunohistochemistry.
Immunohistochemistry (frozen sections):
A 5-10 μg/mL (APAAP) concentration of a previous lot was used in immunohistochemistry.
Immunohistochemistry:
The clone 12H11 works best in fresh frozen or acetone fixed human tissues, however some groups have had reactivity in traditional formalin fixed tissue when the tissue is of human origin. It is recommended that ABC or enhanced detection systems be employed for the best visualization in either acetone or formalin fixed tissues. The antibody reacts with human tissues best. Rat tissue will also react but at a lower affinity, and the antibody does not react with mouse, other species have not been examined. Microwave citric acid buffer or trypsin digestion antigen recovery have both been successful with 12H11 on formalin fixed tissues.
Western blot: 5-10 µg/mL (ECL)
Electrophoretic Mobility Supershift Assay:
A 0.5-1 μg/mL concentration of a previous lot was used in Supershift assay.
Flow cytometry:
A previous lot of this antibody was used in flow cytometry. Fixed cells only, acetone fixed cells.
Optimal working dilutions must be determined by end user.
Control:
TNF α-treated HeLa cells, PMA and calcium ionophore-treated Jurkat cells.
Quality Assurance:
Routinely evaluated by Western Blot on PC12 lysates.
Western blot:
1:500 dilution of this lot detected NF KAPPA B, P65 on 10 μg of PC12 lysates.
Western blot:
1:500 dilution of this lot detected NF KAPPA B, P65 on 10 μg of PC12 lysates.
Purification Method:
Protein A purfied
Presentation:
Purified mouse monoclonal IgG3 liquid in buffer containing 0.02 M Phosphate buffer, 0.25 M NaCl, pH 7.6 with 0.1% sodium azide.
Storage Conditions:
Stable for 6 months at 2-8ºC from date of receipt.
Aliquot to avoid repeated freezing and thawing. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
Aliquot to avoid repeated freezing and thawing. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
UniProt Number:
Entrez Gene Number:
Gene Symbol:
- RELA
- NFKB
- p65
- p50/p65
Alternate Names:
Rel A
Usage Statement:
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
Key Applications:
- Electrophoretic Mobility Shift Assay
- Flow Cytometry
- Immunocytochemistry
- Immunofluorescence
- Immunohistochemistry
- Immunohistochemistry (Paraffin)
- Western Blotting
Entrez Gene Summary:
The p50 (NFKB1)/p65 (RELA) heterodimer is the most abundant form of NFKB. The NFKB complex is inhibited by I-kappa-B proteins (NFKBIA, MIM 164008 or NFKBIB, MIM 604495), which inactivate NFKB by trapping it in the cytoplasm. Phosphorylation of serine residues on the I-kappa-B proteins by kinases (IKBKA, MIM 600664, or IKBKB, MIM 603258) marks them for destruction via the ubiquitination pathway, thereby allowing activation of the NFKB complex. Activated NFKB complex translocates into the nucleus and binds DNA at kappa-B-binding motifs such as 5-prime GGGRNNYYCC 3-prime or 5-prime HGGARNYYCC 3-prime (where H is A, C, or T; R is an A or G purine; and Y is a C or T pyrimidine).
UniProt Summary:
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.
Subunit structure: Component of the NF-kappa-B p65-p50 complex. Component of the NF-kappa-B p65-c-Rel complex. Homodimer; component of the NF-kappa-B p65-p65 complex. Component of the NF-kappa-B p65-p52 complex. May interact with ETHE1. Binds AES and TLE1. Interacts with TP53BP2. Binds to and is phosphorylated by the activated form of either RPS6KA4 or RPS6KA5. Interacts with ING4 and this interaction may be indirect. Interacts with CARM1, USP48 and UNC5CL. Interacts with IRAK1BP1. Interacts with NFKBID. Interacts with NFKBIA. Interacts with GSK3B. Interacts with NFKBIB. Interacts with NFKBIE. Interacts with NFKBIZ. Part of a 70-90 kDa complex at least consisting of CHUK, IKBKB, NFKBIA, RELA, IKBKAP and MAP3K14. Interacts with HDAC3; HDAC3 mediates the deacetylation of RELA. Interacts with HDAC1; the interaction requires non-phosphorylated RELA. Interacts with CBP; the interaction requires phosphorylated RELA. Interacts (phosphorylated at 'Thr-254') with PIN1; the interaction inhibits p65 binding to NFKBIA. Interacts with SOCS1. Interacts with UXT. Interacts with MTDH and PHF11. Interacts with ARRB2. Interacts with human respiratory syncytial virus (HRSV) protein M2-1. Interacts with NFKBIA (when phosphorylated), the interaction is direct; phosphorylated NFKBIA is part of a SCF(BTRC)-like complex lacking CUL1.
Subcellular location: Nucleus. Cytoplasm. Note: Nuclear, but also found in the cytoplasm in an inactive form complexed to an inhibitor (I-kappa-B).
Post-translational modification: Ubiquitinated, leading to its proteosomal degradation. Degradation is required for termination of NF-kappa-B response.
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.
Sequence similarities: Contains 1 RHD (Rel-like) domain.
Subunit structure: Component of the NF-kappa-B p65-p50 complex. Component of the NF-kappa-B p65-c-Rel complex. Homodimer; component of the NF-kappa-B p65-p65 complex. Component of the NF-kappa-B p65-p52 complex. May interact with ETHE1. Binds AES and TLE1. Interacts with TP53BP2. Binds to and is phosphorylated by the activated form of either RPS6KA4 or RPS6KA5. Interacts with ING4 and this interaction may be indirect. Interacts with CARM1, USP48 and UNC5CL. Interacts with IRAK1BP1. Interacts with NFKBID. Interacts with NFKBIA. Interacts with GSK3B. Interacts with NFKBIB. Interacts with NFKBIE. Interacts with NFKBIZ. Part of a 70-90 kDa complex at least consisting of CHUK, IKBKB, NFKBIA, RELA, IKBKAP and MAP3K14. Interacts with HDAC3; HDAC3 mediates the deacetylation of RELA. Interacts with HDAC1; the interaction requires non-phosphorylated RELA. Interacts with CBP; the interaction requires phosphorylated RELA. Interacts (phosphorylated at 'Thr-254') with PIN1; the interaction inhibits p65 binding to NFKBIA. Interacts with SOCS1. Interacts with UXT. Interacts with MTDH and PHF11. Interacts with ARRB2. Interacts with human respiratory syncytial virus (HRSV) protein M2-1. Interacts with NFKBIA (when phosphorylated), the interaction is direct; phosphorylated NFKBIA is part of a SCF(BTRC)-like complex lacking CUL1.
Subcellular location: Nucleus. Cytoplasm. Note: Nuclear, but also found in the cytoplasm in an inactive form complexed to an inhibitor (I-kappa-B).
Post-translational modification: Ubiquitinated, leading to its proteosomal degradation. Degradation is required for termination of NF-kappa-B response.
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.
Sequence similarities: Contains 1 RHD (Rel-like) domain.
Brand Family:
Chemicon
Protein/Isoform Description:
The transcription factor NFκB (Nuclear Factor kappa B) is involved in the expression and regulation of a number of important cellular and physiological processes such as growth, development, apoptosis, immune and inflammatory response. NFκB represents a group of 5 structurally related and evolutionarily conserved proteins in mammals that include Rel (cRel), RelA (p65), RelB, NFκB1 (p50 and its precursor p105), and NFκB2 (p52 and its precursor p100). NFκB/Rel proteins exist as homo- or heterodimers to form transcriptionally competent or repressive complexes, though the p50/p65 heterodimer of NFκB appears to be the most abundant in cells. A critical component in NFκB regulation is the IκB Kinase (IKK) complex. NFκB transcription factors exist in their inactive form and are retained in the cytoplasm by the bound inhibitory IκB proteins. Upon stimulation by multiple inducers including viruses or cytokines, IκB is rapidly phosphorylated and degraded, resulting in the release of the NFκB complex. The p105 subunit is cleaved into its active p50 form. This cleavage exposes the NLS sequence on the p50 subunit. The p50/p65 heteroduplex then translocates to the nucleus where it activates gene transcription. The wide variety of genes regulated by NFκB includes those encoding cytokines, chemokines, adhesion molecules, acute phase proteins and inducible effector enzymes.
Product Name:
Anti-NFκB, p65 subunit, active subunit, clone 12H11
Concentration:
1 mg/mL
Antibody Type:
Monoclonal Antibody
Qty/Pk:
100 µg
Format:
Purified
Host:
Mouse