Key features and details
- One-wash 90 minute protocol
- Sensitivity: 500 pg/ml
- Sample type: Cell Lysate, Tissue Homogenate
- Detection method: Colorimetric
- Assay type: Semi-quantitative
- Reacts with: Mouse, Human
Product nameNF kappaB p65 (pS536 + Total) ELISA Kit
Intra-assay Sample n Mean SD CV% (pS536) 6 3.4% (Total) 6 2.8% Inter-assay Sample n Mean SD CV% (pS536) 3 10.3% (Total) 3 8.7%
Sample typeCell Lysate, Tissue Homogenate
Assay time1h 30m
Assay durationOne step assay
Species reactivityReacts with: Mouse, Human
Predicted to work with: Rat
Abcam’s NFkB p65 (pS536) and NFkB p65 (Total) in vitro SimpleStep ELISA™ (Enzyme-Linked Immunosorbent Assay) kit is designed for the semi-quantitative measurement of NFkB p65 (pS536) and Total NFkB p65 protein in Human and mouse cells.
The SimpleStep ELISA™ employs an affinity tag labeled capture antibody and a reporter conjugated detector antibody which immunocapture the sample analyte in solution. This entire complex (capture antibody/analyte/detector antibody) is in turn immobilized via immunoaffinity of an anti-tag antibody coating the well. To perform the assay, samples or standards are added to the wells, followed by the antibody mix. After incubation, the wells are washed to remove unbound material. TMB substrate is added and during incubation is catalyzed by HRP, generating blue coloration. This reaction is then stopped by addition of Stop Solution completing any color change from blue to yellow. Signal is generated proportionally to the amount of bound analyte and the intensity is measured at 450 nm. Optionally, instead of the endpoint reading, development of TMB can be recorded kinetically at 600 nm.
As of October 2019, this kit was reformulated with new antibodies to maintain continued long term supply.
Estimated sensitivity: Phospho-NF-κB p65 (Ser536): 5 µg/mL (tested with HeLa), Total NF-κB p65: 5 µg/mL (tested with HeLa)
Range: Phospho-NF-κB p65 (Ser536): 6-600 µg/mL, Total NF-κB p65: 6-600 µg/mL
Abcam has not and does not intend to apply for the REACH Authorisation of customers’ uses of products that contain European Authorisation list (Annex XIV) substances.
It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses.
Storage instructionsStore at +4°C. Please refer to protocols.
Components 1 x 96 tests 10X Wash Buffer PT 1 x 15ml 50X Cell Extraction Enhancer Solution 1 x 1ml 5X Cell Extraction Buffer PTR 1 x 12ml Lyophilized NF kappaB p65 Control Lysate 1 vial NF kappaB p65 (pS536) Capture Antibody 1 x 1.5ml NF kappaB p65 (pS536) Detector Antibody 1 x 1.5ml NF kappaB p65 (Total) Capture Antibody 1 x 1.5ml NF kappaB p65 (Total) Detector Antibody 1 x 1.5ml Plate Seal 1 unit SimpleStep Pre-Coated 96-Well Microplate (ab206978) 1 unit Stop Solution 1 x 12ml TMB Substrate 1 x 12ml
Cellular localizationNF-kB p65: Nucleus. Cytoplasm. Nuclear, but also found in the cytoplasm in an inactive form complexed to an inhibitor (I-kappa-B). Colocalized with RELA in the nucleus upon TNF-alpha induction. NF-kappaB p65 (Total): Nucleus. Cytoplasm. Nuclear, but also found in the cytoplasm in an inactive form complexed to an inhibitor (I-kappa-B). Colocalized with RELA in the nucleus upon TNF-alpha induction.
SimpleStep ELISA technology allows the formation of the antibody-antigen complex in one single step, reducing assay time to 90 minutes. Add samples or standards and antibody mix to wells all at once, incubate, wash, and add your final substrate. See protocol for a detailed step-by-step guide.
Example of a typical NFĸB p65 (pS536) and NFĸB p65 (Total) cell lysate dilution series. Raw data values are shown in the table. Background-subtracted data values (mean +/- SD) are graphed.
Linearity of dilution in representative sample matrices. Cellular lysates were prepared at 3 concentrations in common media containing 1X Cell Extraction Buffer PTR. Data from duplicate measurements of NFĸB p65 (pS536) are normalized and plotted.
Cell line analysis for Total NFĸB p65 from 200 µg/mL preparations of cell extracts. Data from triplicate measurements (mean +/- SD) are plotted and compared to 1X Cell Extraction Buffer PTR (zero).
Induction of NFĸB p65 (pS536) phosphorylation in MCF-7 cells in response to TNFα treatment. HeLa cells were cultured in 96-well tissue culture plates, and treated (10 min) with a dose-range of TNFα before cell lysis. Data from quadruplicate measurements of NFĸB p65 (pS536) are plotted and compared against total NFĸB p65 protein levels. Comparative NFĸB p65 (pS536) and NFĸB p65 (Total) data also shown by Western Blot.
ab176663 has been referenced in 6 publications.
- Saber S et al. Telmisartan attenuates N-nitrosodiethylamine-induced hepatocellular carcinoma in mice by modulating the NF-?B-TAK1-ERK1/2 axis in the context of PPAR? agonistic activity. Naunyn Schmiedebergs Arch Pharmacol N/A:N/A (2019). PubMed: 31367864
- Torbica T et al. Chronic Inflammation in Response to Injury: Retention of Myeloid Cells in Injured Tissue Is Driven by Myeloid Cell Intrinsic Factors. J Invest Dermatol 139:1583-1592 (2019). PubMed: 30703358
- Alrdahe S et al. Dysregulation of macrophage development and phenotype in diabetic human macrophages can be rescued by Hoxa3 protein transduction. PLoS One 14:e0223980 (2019). PubMed: 31626638
- Al-Dwairi A et al. Metformin exerts anti-inflammatory effects on mouse colon smooth muscle cells in vitro. Exp Ther Med 16:985-992 (2018). PubMed: 30116349
- Song L et al. c-Kit modifies the inflammatory status of smooth muscle cells. PeerJ 5:e3418 (2017). ELISA ; Mouse . PubMed: 28626608
- Kroetz DN et al. Type I Interferon Induced Epigenetic Regulation of Macrophages Suppresses Innate and Adaptive Immunity in Acute Respiratory Viral Infection. PLoS Pathog 11:e1005338 (2015). Sandwich ELISA ; Human . PubMed: 26709698