PhosphoTracer NF-kappaB p65 Total ELISA Kit (ab119637)
- Product namePhosphoTracer NF-kappaB p65 Total ELISA Kit
- Detection methodFluorescent
- Tests1 x 96 well plate
- Sample typeCell culture extracts
- Assay typeSandwich
- Sensitivity10 µg protein/ml
- Range10 µg protein/ml - 1000 µg protein/ml
- Assay time1h 15m
- Assay durationOne step assay
- Species reactivityReacts with: Mouse, Human
Predicted to work with: Rat
- Product overview
PhosphoTracer assays use a traditional immuno-sandwich format, but with a major difference both the analyte and the assay reagents are added to the PhosphoTracer assay microplate at the same time. After a short incubation period, unbound assay reagents and analytes are washed away, and immuno-complexes containing both antibodies are detected. The process can take as little as 60 minutes to complete.
PhosphoTracer kits also allow a higher degree of assay flexibility. In contrast to other ELISA formats, no antibodies are present on the assay microplate itself, so assays for several different targets can be performed in different wells on the same microplate. Simply mix the lysate with your target reagents of choice, using the microplate configuration of your choice.
A whole new way of performing cellular assays, PhosphoTracer takes the hard work out of running a standard ELISA, while still giving the high quality results expected from a sandwich immunoassay. Fully self-contained kits are supplied in convenient 96-well packs. Simple to use and highly sensitive PhosphoTracer kits are designed to get results, fast.
Abcam’s PhosphoTracer NF-kappaB p65 assay kits detect endogenous levels of NF-kappaB p65 (GenBank Accession NP_068810) in cellular lysates. Total NF-kappaB p65 assay kits detect NF-kappaB p65 irrespective of phosphorylation status.
The substrate used with the HRP conjugated detection antibody is a combination of 10-Acetyl-3,7-dihydroxyphenoxazine (ADHP) (wavelength exc/em = 530-540nm / 590-600nm), a highly sensitive and stable substrate for HRP) and ADHP Dilution Buffer (a stabilized H2O2 solution). Learn more about the fluorogenic substrate, ADHP.
- Tested applicationsSandwich ELISA more details
- Storage instructionsStore at +4°C. Please refer to protocols.
Components 1 x 96 tests 96-well PhosphoTracer assay plate (stripwell) 1 unit Adherent plate seal 2 units ADHP (100X) 1 x 120µl ADHP Dilution Buffer 1 x 15ml Assay Control Lysate (lyophilized) 1 x 0.25ml Enhancer Solution 1 x 1ml Lysis Buffer (5X) 1 x 15ml Mouse monoclonal NFkappaB p65 (HRP) (96 assay points) 1 x 3ml Rabbit monoclonal NFkappaB p65 (96 assay points) 1 x 3ml Stop Solution 1 x 2ml Wash Buffer (10X) 1 x 15ml
- Research Areas
modificationsUbiquitinated, leading to its proteasomal degradation. Degradation is required for termination of NF-kappa-B response.
Monomethylated at Lys-310 by SETD6. Monomethylation at Lys-310 is recognized by the ANK repeats of EHMT1 and promotes the formation of repressed chromatin at target genes, leading to down-regulation of NF-kappa-B transcription factor activity. Phosphorylation at Ser-311 disrupts the interaction with EHMT1 without preventing monomethylation at Lys-310 and relieves the repression of target genes.
Phosphorylation at Ser-311 disrupts the interaction with EHMT1 and promotes transcription factor activity (By similarity). 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. Interaction with BRMS1 promotes deacetylation of 'Lys-310'.
- NF kappa B p65delta3NFKB, p65 subunitNFKB3
- Nuclear factor kappa-B, subunit 3Nuclear factor NF-kappa-B p65 subunitNuclear factor of kappa light chain gene enhancer in B cells3Nuclear factor of kappa light polypeptide gene enhancer in B-cells 3p65relATF65_HUMANTranscription factor NFKB3Transcription factor p65V rel reticuloendotheliosis viral oncogene homolog AV rel reticuloendotheliosis viral oncogene homolog A (avian)
Our Abpromise guarantee covers the use of ab119637 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|Sandwich ELISA||sELISA. Tested in HeLa, THP1, Jurkat|
PhosphoTracer NF-kappaB p65 Total ELISA Kit images
Detection of total NF-kappaB p65, using ab119637 on HeLa cells stimulated with various concentrations of TNF alpha.
Using the PhosphoTracer Total NF-kappaB p65 assay, the amount of lysate required to detect NF-kappaB p65 in TNFalpha-treated HeLa cell lysates was examined. Up to 50 μg of lysate/well was loaded into replicate wells of a PhosphoTracer microplate, and analysed for total NF-kappaB p65. Total NF-kappaB p65 was readily detected in less than 10 μg of lysate/well.
Using the NF-kappaB p65 assay kits, a significant stimulation of NF-kappaB p65 phosphorylation at Ser536 is detected in HeLa cells treated with TNFalpha for 10 minutes (+) compared with untreated cells (-), while no change in total NF-kappaB p65 levels is observed.
Using Western blot, a significant stimulation of NF-kappaB p65 phosphorylation at Ser536 is detected in HeLa cells treated with TNFalpha for 10 minutes (+) compared with untreated cells (-), while no change in total NF-kappaB p65 levels is observed.
References for PhosphoTracer NF-kappaB p65 Total ELISA Kit (ab119637)
ab119637 has not yet been referenced specifically in any publications.