The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/10000 - 1/50000. Detects a band of approximately 110 kDa (predicted molecular weight: 97 kDa).
1/10 - 1/100.
Application notesIs unsuitable for Flow Cyt or IHC-P.
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. 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.
Involvement in diseaseNote=A chromosomal aberration involving NFKB2 is found in a case of B-cell non Hodgkin lymphoma (B-NHL). Translocation t(10;14)(q24;q32) with IGHA1. The resulting oncogene is also called Lyt-10C alpha variant. Note=A chromosomal aberration involving NFKB2 is found in a cutaneous T-cell leukemia (C-TCL) cell line. This rearrangement produces the p80HT gene which encodes for a truncated 80 kDa protein (p80HT). Note=In B-cell leukemia (B-CLL) cell line, LB40 and EB308, can be found after heterogeneous chromosomal aberrations, such as internal deletions.
DomainThe C-terminus of p100 might be involved in cytoplasmic retention, inhibition of DNA-binding by p52 homodimers, and/or transcription activation. The glycine-rich region (GRR) appears to be a critical element in the generation of p52.
Post-translational modificationsWhile translation occurs, the particular unfolded structure after the GRR repeat promotes the generation of p52 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. Subsequent to MAP3K14-dependent serine phosphorylation, p100 polyubiquitination occurs then triggering its proteasome-dependent processing. Constitutive processing is tightly suppressed by its C-terminal processing inhibitory domain, named PID, which contains the death domain.
Cellular localizationNucleus. Cytoplasm. Nuclear, but also found in the cytoplasm in an inactive form complexed to an inhibitor.
Lymphocyte translocation chromosome 10 protein antibody
Lyt 10 antibody
NF kB2 antibody
Nuclear factor NF kappa B p100 subunit antibody
Nuclear factor NF kappa B p52 subunit antibody
Nuclear factor NF-kappa-B p52 subunit antibody
Nuclear factor of kappa light chain gene enhancer in B cells 2 antibody
Nuclear factor of kappa light polypeptide gene enhancer in B cells 2 antibody
Nuclear factor of kappa light polypeptide gene enhancer in B-cells 2 antibody
Oncogene Lyt 10 antibody
Oncogene Lyt-10 antibody
Anti-NFkB p100 / p52 antibody [EPR4686] images
Western blot - Anti-NFkB p100 / p52 antibody [EPR4686] (ab109440)
Predicted band size : 97 kDa
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.
Western blot - NFkB p100 / p52 antibody [EPR4686] (ab109440)
All lanes : Anti-NFkB p100 / p52 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