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Mouse E1A Binding Protein P300 (EP300) ELISA Kit

Principle of the Assay

The microtiter plate provided in this kit has been pre-coated with an antibody specific to EP300. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific to EP300. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After the TMB substrate solution is added, only those wells that contain EP300, biotin-conjugated antibody, and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution, and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of EP300 in the samples is then determined by comparing the O.D. of the samples to the standard curve.


For Use with serum, plasma, and cell culture supernatants. For Research Use Only. Not for use in diagnostic procedures.

Target Information

Functions as histone acetyltransferase and regulates transcription via chromatin remodeling (By similarity). Acetylates all four core histones in nucleosomes (By similarity). Histone acetylation gives an epigenetic tag for transcriptional activation (By similarity). Mediates cAMP-gene regulation by binding specifically to phosphorylated CREB protein (PubMed:18486321, PubMed:24216764). Mediates acetylation of histone H3 at 'Lys-122' (H3K122ac), a modification that localizes at the surface of the histone octamer and stimulates transcription, possibly by promoting nucleosome instability (By similarity). Mediates acetylation of histone H3 at 'Lys-27' (H3K27ac) (By similarity). Also functions as acetyltransferase for non-histone targets, such as ALX1, HDAC1, PRMT1 or SIRT2 (PubMed:28883095, PubMed:28576496). Acetylates 'Lys-131' of ALX1 and acts as its coactivator (By similarity). Acetylates SIRT2 and is proposed to indirectly increase the transcriptional activity of TP53 through acetylation and subsequent attenuation of SIRT2 deacetylase function (By similarity). Following DNA damage, forms a stress-responsive p53/TP53 coactivator complex with JMY which mediates p53/TP53 acetylation, thereby increasing p53/TP53-dependent transcription and apoptosis (By similarity). Promotes chromatin acetylation in heat shock responsive HSP genes during the heat shock response (HSR), thereby stimulating HSR transcription (By similarity). Acetylates HDAC1 leading to its inactivation and modulation of transcription (By similarity). Acetylates 'Lys-247' of EGR2 (PubMed:28576496). Acts as a TFAP2A-mediated transcriptional coactivator in presence of CITED2 (By similarity). Plays a role as a coactivator of NEUROD1-dependent transcription of the secretin and p21 genes and controls terminal differentiation of cells in the intestinal epithelium (By similarity). Promotes cardiac myocyte enlargement (By similarity). Can also mediate transcriptional repression (By similarity). Acetylates FOXO1 and enhances its transcriptional activity (By similarity). Acetylates BCL6 wich disrupts its ability to recruit histone deacetylases and hinders its transcriptional repressor activity (By similarity). Participates in CLOCK or NPAS2-regulated rhythmic gene transcription; exhibits a circadian association with CLOCK or NPAS2, correlating with increase in PER1/2 mRNA and histone H3 acetylation on the PER1/2 promoter (By similarity). Acetylates MTA1 at 'Lys-626' which is essential for its transcriptional coactivator activity (PubMed:14645221, PubMed:9512516). Acetylates XBP1 isoform 2; acetylation increases protein stability of XBP1 isoform 2 and enhances its transcriptional activity (PubMed:20955178). Acetylates PCNA; acetylation promotes removal of chromatin-bound PCNA and its degradation during nucleotide excision repair (NER) (By similarity). Acetylates MEF2D (By similarity). Acetylates and stabilizes ZBTB7B protein by antagonizing ubiquitin conjugation and degradation, this mechanism may be involved in CD4/CD8 lineage differentiation (PubMed:20810990). Acetylates GABPB1, impairing GABPB1 heterotetramerization and activity (PubMed:25200183). Acetylates PCK1 and promotes PCK1 anaplerotic activity (By similarity). Acetylates RXRA and RXRG (By similarity). Acetylates isoform M2 of PKM (PKM2), promoting its homodimerization and conversion into a protein kinase (By similarity). In addition to protein acetyltransferase, can use different acyl-CoA substrates, such as (2E)-butenoyl-CoA (crotonyl-CoA), butanoyl-CoA (butyryl-CoA), 2-hydroxyisobutanoyl-CoA (2-hydroxyisobutyryl-CoA), lactoyl-CoA or propanoyl-CoA (propionyl-CoA), and is able to mediate protein crotonylation, butyrylation, 2-hydroxyisobutyrylation, lactylation or propionylation, respectively (PubMed:27105113). Acts as a histone crotonyltransferase; crotonylation marks active promoters and enhancers and confers resistance to transcriptional repressors. Histone crotonyltransferase activity is dependent on the concentration of (2E)-butenoyl-CoA (crotonyl-CoA) substrate and such activity is weak when (2E)-butenoyl-CoA (crotonyl-CoA) concentration is low (By similarity). Also acts as a histone butyryltransferase; butyrylation marks active promoters (PubMed:27105113). Catalyzes histone lactylation in macrophages by using lactoyl-CoA directly derived from endogenous or exogenous lactate, leading to stimulates gene transcription (By similarity). Acts as a protein-lysine 2-hydroxyisobutyryltransferase; regulates glycolysis by mediating 2-hydroxyisobutyrylation of glycolytic enzymes. Functions as a transcriptional coactivator for SMAD4 in the TGF-beta signaling pathway (By similarity).

GENE ID 328572
SWISS PROT B2RWS6
SYNONYMS KAT3B; p300; Histone Acetyltransferase p300


Materials Supplied

Kit Components 96 Wells Quantity/Size
Pre-coated, ready-to-use 96-well strip plate 1 plate
Plate sealer for 96 wells 2
Standard
2 tubes
Diluent buffer 1 bottle
Detection Reagent A 1 bottle
Detection Reagent B 1 bottle
TMB Substrate 1 tube
Stop Solution 1 tube
Wash Buffer (30 ℅ concentrate) 1 tube
Product data sheet 1 copy

Storage

Storage The TMB Substrate, Wash Buffer (30X concentrate), and the Stop Solution should be stored at 4°C upon receipt, while the other items should be stored at -20°C.

Performance Characteristics

REPEATABILITY

Intra-assay Precision (Precision within an assay): 3 samples with low, middle, and high-level EP300 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle, and high-level EP300 were tested on 3 different plates, with 8 replicates in each plate.
CV(%) = SD/meanX100

Intra-Assay: CV<10%
Inter-Assay: CV<12%

SENSITIVITY The minimum detectable dose was 0.051ng/mL.
ASSAY RANGE 0.156-10ng/mL
SPECIFICITY This assay has high sensitivity and excellent specificity for the detection of EP300.
No significant cross-reactivity or interference between EP300 and analogs was observed.
Note:
Limited by current skills and knowledge, it is impossible to perform all possible cross-reactivity detection tests between EP300 and all analogs, therefore, cross reactivity may still exist.