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    kat3bp300-inhibitor-screening-assay-kit-fluorometric-ab196996.pdf

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Epigenetics and Nuclear Signaling Chromatin Modifying Enzymes Acetylation
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KAT3B/p300 Inhibitor Screening Assay Kit (Fluorometric) (ab196996)

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  • Protocol Booklet
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Functional Studies - KAT3B/p300 Inhibitor Screening Assay Kit (Fluorometric) (ab196996)

    Key features and details

    • Assay type: Enzyme activity
    • Detection method: Fluorescent
    • Platform: Microplate reader
    • Sample type: Inhibitor compounds

    Overview

    • Product name

      KAT3B/p300 Inhibitor Screening Assay Kit (Fluorometric)
    • Detection method

      Fluorescent
    • Sample type

      Inhibitor compounds
    • Assay type

      Enzyme activity
    • Product overview

      KAT3B/p300 Inhibitor Screening Assay Kit (Fluorometric) (ab196996) is an assay where histone H3 peptide and Acetyl CoA are used as acetylation substrates. KAT3B/p300 acetylates the Histone H3 peptide and generates Coenzyme A with a free thiol group (CoA-SH). CoA-SH reacts subsequently with a Thiol Detecting Probe in order to increase the amount of fluorescence in the solution, which can be measured at Ex/Em = 392/482 nm. In the presence of KAT2B/p300 specific inhibitors, the enzymatic activity is reduced ot completely abolished resulting in decreased or total loss of fluorescence.

      This assay kit is a simple, sensitive and rapid tool to screen potential p300 inhibitors.

    • Notes

      p300 or KAT3B, a histone acetyltransferase, contributes to transcriptional activation by acetylating chromatin on the lysine residues of H3 and H4 histones. There is growing evidence that p300 plays an important role in cancer cell proliferation and differentiation. p300 inhibitors have potential applications in cancer therapy.

      KTA3B (K-(lysine) acetyltransferase 3B, EC 2.3.1.32), also known as p300 possesses intrinsic histone acetyltransferase (HAT) activity and is able to acetylate lysine residues present in both histone H3 and histone H4.

      There is growing evidence taht p300 plays an important role in cancer cell proliferation and differentiation. p300 inhibitors have potential applications in cancer therapy.

    • Platform

      Microplate reader

    Properties

    • Storage instructions

      Store at -80°C. Please refer to protocols.
    • Components 100 tests
      Acetyl CoA 1 vial
      H3 Peptide 1 vial
      p300 Assay Buffer 1 x 20ml
      p300 Enzyme 1 x 0.1ml
      p300 Inhibitor 1 x 10µl
      Thiol Detecting Probe 1 x 0.2ml
    • Research areas

      • Epigenetics and Nuclear Signaling
      • Chromatin Modifying Enzymes
      • Acetylation
      • Epigenetics and Nuclear Signaling
      • Transcription
      • Other factors
      • Stem Cells
      • Signaling Pathways
      • TGF beta
      • Nuclear
      • Epigenetics and Nuclear Signaling
      • Chromatin Modifying Enzymes
      • Acetylation
      • HAT
      • Cancer
      • Oncoproteins/suppressors
      • Tumor suppressors
      • Rb family
      • Cancer
      • Cancer Metabolism
      • Response to hypoxia
      • Metabolism
      • Pathways and Processes
      • Metabolism processes
      • Hypoxia
      • Metabolism
      • Types of disease
      • Obesity
    • Function

      Functions as histone acetyltransferase and regulates transcription via chromatin remodeling. Acetylates all four core histones in nucleosomes. Histone acetylation gives an epigenetic tag for transcriptional activation. Mediates cAMP-gene regulation by binding specifically to phosphorylated CREB protein. 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. Mediates acetylation of histone H3 at 'Lys-27' (H3K27ac). Also functions as acetyltransferase for nonhistone targets. Acetylates 'Lys-131' of ALX1 and acts as its coactivator. Acetylates SIRT2 and is proposed to indirectly increase the transcriptional activity of TP53 through acetylation and subsequent attenuation of SIRT2 deacetylase function. Acetylates HDAC1 leading to its inactivation and modulation of transcription. Acts as a TFAP2A-mediated transcriptional coactivator in presence of CITED2. 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. Promotes cardiac myocyte enlargement. Can also mediate transcriptional repression. Binds to and may be involved in the transforming capacity of the adenovirus E1A protein. In case of HIV-1 infection, it is recruited by the viral protein Tat. Regulates Tat's transactivating activity and may help inducing chromatin remodeling of proviral genes. Acetylates FOXO1 and enhances its transcriptional activity. Acetylates BCL6 wich disrupts its ability to recruit histone deacetylases and hinders its transcriptional repressor activity. 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. Acetylates MTA1 at 'Lys-626' which is essential for its transcriptional coactivator activity (PubMed:10733570, PubMed:11430825, PubMed:11701890, PubMed:12402037, PubMed:12586840, PubMed:12929931, PubMed:14645221, PubMed:15186775, PubMed:15890677, PubMed:16617102, PubMed:16762839, PubMed:18722353, PubMed:18995842, PubMed:23415232, PubMed:23911289, PubMed:23934153, PubMed:8945521). 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) (PubMed:24939902). Acetylates MEF2D.
    • Involvement in disease

      Defects in EP300 may play a role in epithelial cancer.
      Chromosomal aberrations involving EP300 may be a cause of acute myeloid leukemias. Translocation t(8;22)(p11;q13) with KAT6A.
      Rubinstein-Taybi syndrome 2
    • Sequence similarities

      Contains 1 bromo domain.
      Contains 1 CBP/p300-type HAT (histone acetyltransferase) domain.
      Contains 1 KIX domain.
      Contains 2 TAZ-type zinc fingers.
      Contains 1 ZZ-type zinc finger.
    • Domain

      The CRD1 domain (cell cycle regulatory domain 1) mediates transcriptional repression of a subset of p300 responsive genes; it can be de-repressed by CDKN1A/p21WAF1 at least at some promoters. It conatins sumoylation and acetylation sites and the same lysine residues may be targeted for the respective modifications. It is proposed that deacetylation by SIRT1 allows sumoylation leading to suppressed activity.
    • Post-translational
      modifications

      Acetylated on Lys at up to 17 positions by intermolecular autocatalysis. Deacetylated in the transcriptional repression domain (CRD1) by SIRT1, preferentially at Lys-1020. Deacetylated by SIRT2, preferentially at Lys-418, Lys-423, Lys-1542, Lys-1546, Lys-1549, Lys-1699, Lys-1704 and Lys-1707.
      Citrullinated at Arg-2142 by PADI4, which impairs methylation by CARM1 and promotes interaction with NCOA2/GRIP1.
      Methylated at Arg-580 and Arg-604 in the KIX domain by CARM1, which blocks association with CREB, inhibits CREB signaling and activates apoptotic response. Also methylated at Arg-2142 by CARM1, which impairs interaction with NCOA2/GRIP1.
      Sumoylated; sumoylation in the transcriptional repression domain (CRD1) mediates transcriptional repression. Desumoylated by SENP3 through the removal of SUMO2 and SUMO3.
      Probable target of ubiquitination by FBXO3, leading to rapid proteasome-dependent degradation.
      Phosphorylated by HIPK2 in a RUNX1-dependent manner. This phosphorylation that activates EP300 happens when RUNX1 is associated with DNA and CBFB. Phosphorylated by ROCK2 and this enhances its activity. Phosphorylation at Ser-89 by AMPK reduces interaction with nuclear receptors, such as PPARG.
    • Cellular localization

      Cytoplasm. Nucleus. In the presence of ALX1 relocalizes from the cytoplasm to the nucleus. Colocalizes with ROCK2 in the nucleus.
    • Target information above from: UniProt accession Q09472 The UniProt Consortium
      The Universal Protein Resource (UniProt) in 2010
      Nucleic Acids Res. 38:D142-D148 (2010) .

      Information by UniProt
    • Alternative names

      • E1A associated protein p300
      • E1A binding protein p300
      • E1A-associated protein p300
      • EP300
      • EP300: E1A binding protein p300
      • EP300_HUMAN
      • Histone acetyltransferase p300
      • KAT3B
      • p300 HAT
      • RSTS2
      see all

    Images

    • Functional Studies - KAT3B/p300 Inhibitor Screening Assay Kit (Fluorometric) (ab196996)
      Functional Studies - KAT3B/p300 Inhibitor Screening Assay Kit (Fluorometric) (ab196996)

      Inhibition of p300 activity by the p300 Inhibitor. Assays were performed following the kit protocol.

    Protocols

    • Protocol Booklet

    Click here to view the general protocols

    Datasheets and documents

    • Datasheet
    • SDS
  • References (1)

    Publishing research using ab196996? Please let us know so that we can cite the reference in this datasheet.

    ab196996 has been referenced in 1 publication.

    • Proto MC  et al. Inhibition of Wnt/ß-Catenin pathway and Histone acetyltransferase activity by Rimonabant: a therapeutic target for colon cancer. Sci Rep 7:11678 (2017). Functional Studies . PubMed: 28916833

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