Chromatin immunoprecipitation analysis of p300 was performed using cross-linked chromatin from 1x10^6 HCT116 colon carcinoma cells treated with serum for 0, 15, and 30 minutes. Immunoprecipitation was performed using a multiplex microplate Matrix ChIP assay (see reference for Matrix ChIP protocol: http://www.ncbi.nlm.nih.gov/pubmed/22098709) with 1.0ul/100ul well volume of a p300 polyclonal antibody (Product # PA1-848). Chromatin aliquots from ~1x10^5 cells were used per ChIP pull-down. Quantitative PCR data were done in quadruplicate using 1ul of eluted DNA in 2ul SYBR real-time PCR reactions containing primers to amplify -15kb upstream of the Egr1 gene or exon-1 or exon-2 of Egr1. PCR calibration curves were generated for each primer pair from a dilution series of sheared total genomic DNA. Quantitation of immunoprecipitated chromatin is presented as signal relative to the total amount of input chromatin. Results represent the mean +/- SEM for three experiments. A schematic representation of the Egr-1 locus is shown above the data where boxes represent exons (black boxes = translated regions, white boxes = untranslated regions), the zigzag line represents an intron, and the straight line represents upstream sequence. Regions amplified by Egr-1 primers are represented by black bars. Data courtesy of the Innovators Program.
|Tested species reactivity||Human|
|Published species reactivity||Human|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Synthetic peptide corresponding to residues G(139) T S G P N Q G P T Q S T(151) C in the nuclear factor binding domain of human p300.|
|Purification||Antigen affinity chromatography|
|Storage buffer||PBS with 1mg/ml BSA|
|Contains||0.05% sodium azide|
|Storage Conditions||-20° C, Avoid Freeze/Thaw Cycles|
|Tested Applications||Dilution *|
|ChIP assay (ChIP)||1-3 ul|
|Western Blot (WB)||1-2 µg/ml|
|ChIP assay (ChIP)||See 1 publications below|
PA1-848 detects p300 from human cells.
PA1-848 has been successfully used in Western blot and ChIP procedures. By Western blot, this antibody detects an ~265 kDa protein representing p300 from HeLa cell lysate.
The PA1-848 immunogen is a synthetic peptide corresponding to residues G(139) T S G P N Q G P T Q S T(151) C in the nuclear factor binding domain of human p300. The PA1-848 immunizing peptide (Cat. # PEP-053) is available for use in neutralization and control experiments.
Cyclic AMP-responsive enhancer binding protein (CREB) binding protein (CBP) and p300 are closely related transcriptional coactivators and have been shown to directly interact with many different DNA-binding transcription factors including nuclear hormone receptors, CREB, c-Fos, c-Jun/v-Jun, c-Myb/v-Myb, TFIIB and MyoD. Both CBP and p300 have been shown to display histone acetyltransferase (HAT) activity, capable of acetylating all four core histone particles in nucleosomes. As a result of HAT activity, it has been suggested CBP and p300 may play a direct role in activating chromatin for transcription.
Single point mutations in CBP have been proposed as causative factors in the developmental abnormalities of Rubinstein-Taybi syndrome (RTS). Although both CBP and p300 appear to function similarly, the inability of p300 to rescue CBP malfunction in RTS suggests intrinsic functional differences between CBP and p300.
G Protein-coupled Receptor 40 (GPR40) and Peroxisome Proliferator-activated Receptor ? (PPAR?): AN INTEGRATED TWO-RECEPTOR SIGNALING PATHWAY.
PA1-848 was used in ChIP assay to investigate GPR40 and PPARgamma signaling interactions in endothelial cells
|Wang S,Awad KS,Elinoff JM,Dougherty EJ,Ferreyra GA,Wang JY,Cai R,Sun J,Ptasinska A,Danner RL||The Journal of biological chemistry (290:19544)||2015|