Anti-Phospho-NFKB p65 (Thr505) Polyclonal Antibody is a Rabbit antibody targeting Phospho-NFKB p65 (Thr505). Anti-Phospho-NFKB p65 (Thr505) Polyclonal Antibody can be used in WB,FCM.

IgG

Human,Mouse (predicted:Rat,Dog,Pig,Cow)

1. Sample:
Molt-4 (Human) Cell Lysate at 30 μg
A549 (Human) Cell Lysate at 30 μg
JurkaT (Human) Cell Lysate at 30 μg
Raw264.7 (Mouse) Cell Lysate at 30 μg
Primary: Anti-phospho-NFKB p65 (Thr505) (TMAB-11044) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 65 kD
Observed band size: 62 kD
2. Sample:
Pancreas (Mouse) Lysate at 40 μg
HL60 (Human) Cell Lysate at 30 μg
Primary: Anti-phospho-NFKB p65 (Thr505) (TMAB-11044) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 65 kD
Observed band size: 65 kD
3. Blank control: HL-60.
Primary Antibody (green line): Rabbit Anti-phospho-NFKB p65 (Thr505) antibody (TMAB-11044)
Dilution: 1 μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG.
Secondary Antibody: Goat anti-rabbit IgG-FITC
Dilution: 1 μg /test.
Protocol
The cells were fixed with 4% PFA (10 min at room temperature) and then permeabilized with 90% ice-cold methanol for 20 min at-20℃. The cells were then incubated in 5% BSA to block non-specific protein-protein interactions for 30 min at room temperature. Cells stained with Primary Antibody for 30 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20,000 events was performed.
4. Sample: Hcclm3 Cell Lysate at 30 μg
Primary: Anti-phospho-NFKB p65 (Thr505) (TMAB-11044) at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 61 kD
Observed band size: 61/58 kD

WB: 1:500-2000; FCM: 1μg/Test

Polyclonal

KLH conjugated synthesised phosphopeptide: human around the phosphorylation site of Thr505

Human

RELA

Transcription factor p65

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 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. Associates with chromatin at the NF-kappa-B promoter region via association with DDX1.