NF kappaB p65 (pS536 + Total) ELISA Kit (ab176663)
- Datasheet
- References (2)
- Protocols
Overview
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Product nameNF kappaB p65 (pS536 + Total) ELISA Kit
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Detection methodColorimetric
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Precision
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
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Assay typeSemi-quantitative
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Sensitivity500 pg/ml
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Assay time1h 30m
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Assay durationOne step assay
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Species reactivityReacts with: Mouse, Human
Predicted to work with: Rat
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Product overview
Abcam’s NF?B p65 (pS536) and NF?B p65 (Total) in vitro SimpleStep ELISA™ (Enzyme-Linked Immunosorbent Assay) kit is designed for the semi-quantitative measurement of NF?B p65 (pS536) and Total NF?B 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.
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Notes
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 -
Tested applicationsSuitable for: Sandwich ELISAmore details
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PlatformMicroplate
Properties
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Storage instructionsStore at +4°C. Please refer to protocols.
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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 (ab193970) 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 -
Research areas
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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.
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Database links
- Entrez Gene: 5970 Human
- Entrez Gene: 19697 Mouse
- Entrez Gene: 19697 Mouse
- Entrez Gene: 309165 Rat
- Entrez Gene: 309165 Rat
- Omim: 164014 Human
- SwissProt: Q04206 Human
- SwissProt: Q04207 Mouse
see all
Applications
Our Abpromise guarantee covers the use of ab176663 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 |
|---|---|---|
| Sandwich ELISA | Use at an assay dependent concentration. |
Images
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Typical cell lysate dilution seriesExample 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.
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Linearity of dilutionLinearity 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.
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Comparison of NF?B p65 expression in different cell linesCell 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).
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NF?B p65 (pS536) phosphorylation in response to TNFa treatmentInduction 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.
Protocols
References
This product has been referenced in:
- Song L et al. c-Kit modifies the inflammatory status of smooth muscle cells. PeerJ 5:e3418 (2017). ELISA ; Mouse . Read more (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 . Read more (PubMed: 26709698) »
