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p300 Tools and Services for Research and Drug Discovery

Active Motif offers a workflow of research tools and approaches to characterize the complex functions of p300/CBP.

p300 and CBP (CREB Binding Protein) are transcriptional co-activators that regulate gene expression through acetylation of histones and chromatin remodeling. p300 interacts with over 50 intracellular proteins affecting a range of signaling pathways, while mutations in p300 can lead to dysregulated cell growth and division, and are implicated in acute myeloid leukemia (AML) and multiple solid tumor types.

p300

 

Featured Publication: Raisner, R., et. al. 2018. “Enhancer Activity Requires CBP/P300 Bromodomain-Dependent Histone H3K27 Acetylation.” Cell Reports 24: 1722-1729.
(Active Motif products used in this study: Recombinant p300 protein, Recombinant CREBBP (CBP), Histone PTM antibodies, and RIME services).

Recombinant p300 proteins – Histone Acetyltransferase Activity with Histone H4 Substrate

Activity of Recombinant p300 proteins was assessed using the Active Motif HAT Assay Kit. Equimolar amounts of p300, Catalytic Domain (Cat. No. 81093) and p300, Full Length (81158) proteins were incubated with H4 peptide for 30 minutes to 6 hours, and developed as per the kit protocol. Graph shows averages ± SEM of arbitrary fluorescence units (AFU).

Recombinant p300 proteins – Histone Acetyltransferase Activity with Histone H3 Substrate

Activity of p300, Catalytic Domain (Cat. No. 81093), p300, Full Length (Cat. No. 81158), and p300, Bromodomain (Cat. No. 31372) was assessed using the Active Motif HAT Assay Kit. Equimolar amounts of p300 proteins were incubated with H3 peptide for 3 hours, and developed as per the kit protocol. Anacardic acid, an inhibitor of HAT activity, was used at 15μM / well. Graph shows averages ± SEM of arbitrary fluorescence units (AFU).

Histone Acetyltransferase Activity of Recombinant p300 Proteins by Western Blot

Recombinant Histone H4 (Cat. No. 31493) was incubated with 0 μg, 0.25 μg or 0.5 μg of p300 proteins in a 30 μl reaction system including 50 mM Tris pH 8.0, 1 mM TCEP, 0.1 mM EDTA, 50 ng/μl BSA and 20 μM acetyl-CoA for 30 minutes at 30°C. For each reaction, 7.5 μl of reaction product was run on a 12.5% SDS-PAGE gel and detected by Western Blot using Histone H4 pan-acetyl antibody (Cat. No. 39243).

p300 antibody (mAb) tested by ChIP-Seq

ChIP was performed using the ChIP-IT® High Sensitivity Kit (Cat. No. 53040) with chromatin from 4.5 million LNCaP cells and 5 µl of p300 antibody (Cat. No. 61401). ChIP DNA was sequenced on the Illumina HiSeq and 15 million sequence tags were mapped to identify p300 binding sites. p300 along with H3K4me1 are markers of active enhancer elements and are therefore expected to co-localize. A sampling of the p300 ChIP-Seq data shows the expected co-localization of p300 and H3K4me1.

Products

Recombinant Proteins 

Name Expressed In Format Cat. No.
Recombinant p300 protein Baculovirus 10 µg 81158
Recombinant p300 protein, catalytic domain Baculovirus 20 µg 81093
1 mg 81893
Recombinant p300 (1041-1161) protein E. coli 100 µg 31372
1 mg 31801
Recombinant CREBBP (1075-1873) protein Baculovirus 20 µg 31590
1 mg 31990
Recombinant CREBBP (1081-1197) protein E. coli 100 µg 31373
1 mg 31873
Recombinant KAT2A (GCN5) protein Baculovirus 20 µg 31591
1 mg 31991
Recombinant KAT2B / PCAF protein Baculovirus 20 µg 81142
1 mg 81842
Recombinant KAT2B / PCAF (715-829) protein E. coli 100 µg 31370
1 mg 31770
Recombinant p53 (TP53) protein Baculovirus 20 µg 81091
1 mg 81791

 

Antibodies

Name Isotype Reactivity Appl. Format Cat. No.
p300 antibody (mAb) IgG1 Human ChIP, ChIP-Seq, ICC, IF, IP, WB 100 µg 61401
10 µg 61402
50 µg 61403
AbFlex® Histone H3K27ac antibody (rAb) IgG2a Human ChIP-Seq, ELISA, WB 100 µg 91193
10 µg 91194
Histone H3K27ac antibody (pAb) IgG Budding Yeast, Human, Other (Wide Range) ChIP, ChIP-Seq, ICC, IF, IP, WB 100 µg 39133
10 µg 39134
50 µg 39034
Histone H3K27ac antibody (mAb) IgG1 Human, Other (Wide Range) ChIP, ChIP-Seq, DB, ICC, IF, WB 100 µg 39685
50 µg 39085
RNA pol II antibody (mAb) IgG2a Human ChIP, ChIP-Seq, ICC, IF, WB 100 µg 61667
10 µg 61668

 

Kits

Name Format Cat. No.
HAT Assay Kit (Fluorescent) 1 x 96 rxns 56100