p300 Colorimetric Cell-Based ELISA Kit (CBCAB00340)

Product Name:	p300 Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00340
ELISA Type:	Cell-Based
Target:	p300
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

The p300 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect p300 protein expression profile in cells. The kit can be used for measuring the relative amounts of p300 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on p300.

Qualitative determination of p300 concentration is achieved by an indirect ELISA format. In essence, p300 is captured by p300-specific primary antibodies while the HRP-conjugated secondary antibodies bind the Fc region of the primary antibody. Through this binding, the HRP enzyme conjugated to the secondary antibody can catalyze a colorimetric reaction upon substrate addition. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:

1.	A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values.
2.	Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted.

Database Information:	Gene ID: 2033, UniProt ID: Q09472, OMIM: 602700, Unigene: Hs.517517/Hs.655211
Gene Symbol:	EP300
Sub Type:	None

UniProt Protein Function:	p300: a histone acetyltransferase and transcriptional co-activator that regulates transcription via chromatin remodeling. Acetylates all four core histones in nucleosomes. Related to CPB (CREB-binding protein), and like CPB can stimulate transcription through activation of CREB. Specifically inhibited by the adenovirus oncoprotein E1A. A co-activator of HIF1A (hypoxia-inducible factor 1 alpha), and thus plays a role in the stimulation of hypoxia-induced genes such as VEGF. Mediates cAMP-gene regulation by binding specifically to phosphorylated CREB protein. Methylated at R580 and R604 in the KIX domain by CARM1, which blocks association with CREB, inhibits CREB signaling and activates the apoptotic response. Also methylated at R2142 by CARM1, which impairs interaction with NCoA2.
UniProt Protein Details:	Protein type:Transcription, coactivator/corepressor; Motility/polarity/chemotaxis; EC 2.3.1.48; Nuclear receptor co-regulator; Acetyltransferase Chromosomal Location of Human Ortholog: 22q13.2 Cellular Component: cytoplasm; histone acetyltransferase complex; nucleoplasm; nucleus; transcription factor complex Molecular Function:acetyltransferase activity; androgen receptor binding; beta-catenin binding; chromatin binding; chromatin DNA binding; damaged DNA binding; DNA binding; histone acetyltransferase activity; lysine N-acetyltransferase activity; nuclear hormone receptor binding; p53 binding; protein binding; protein C-terminus binding; transcription activator binding; transcription coactivator activity; transcription factor binding; transferase activity, transferring acyl groups; zinc ion binding Biological Process: apoptosis; B cell differentiation; circadian rhythm; DNA damage response, signal transduction by p53 class mediator resulting in induction of apoptosis; DNA repair; establishment and/or maintenance of chromatin architecture; fat cell differentiation; G2/M transition of mitotic cell cycle; gene expression; heart development; innate immune response; internal peptidyl-lysine acetylation; internal protein amino acid acetylation; lung development; mitotic cell cycle; N-terminal peptidyl-lysine acetylation; negative regulation of transcription from RNA polymerase II promoter; nervous system development; Notch signaling pathway; nucleotide-excision repair; organ morphogenesis; platelet formation; positive regulation of gene expression, epigenetic; positive regulation of interferon type I production; positive regulation of protein binding; positive regulation of transcription factor activity; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription of target genes involved in unfolded protein response; protein amino acid acetylation; protein stabilization; regulation of cell cycle; regulation of gene expression, epigenetic; regulation of transcription, DNA-dependent; response to estrogen stimulus; response to hypoxia; skeletal muscle development; somitogenesis; stimulatory C-type lectin receptor signaling pathway; transcription from RNA polymerase II promoter; transcription-coupled nucleotide-excision repair; viral reproduction Disease: Colorectal Cancer; Rubinstein-taybi Syndrome 1; Rubinstein-taybi Syndrome 2
NCBI Summary:	This gene encodes the adenovirus E1A-associated cellular p300 transcriptional co-activator protein. It functions as histone acetyltransferase that regulates transcription via chromatin remodeling and is important in the processes of cell proliferation and differentiation. It mediates cAMP-gene regulation by binding specifically to phosphorylated CREB protein. This gene has also been identified as a co-activator of HIF1A (hypoxia-inducible factor 1 alpha), and thus plays a role in the stimulation of hypoxia-induced genes such as VEGF. Defects in this gene are a cause of Rubinstein-Taybi syndrome and may also play a role in epithelial cancer. [provided by RefSeq, Jul 2008]
UniProt Code:	Q09472
NCBI GenInfo Identifier:	223590203
NCBI Gene ID:	2033
NCBI Accession:	Q09472.2
UniProt Secondary Accession:	Q09472,B1AKC2,
UniProt Related Accession:	Q09472
Molecular Weight:	264,161 Da
NCBI Full Name:	Histone acetyltransferase p300
NCBI Synonym Full Names:	E1A binding protein p300
NCBI Official Symbol:	EP300
NCBI Official Synonym Symbols:	p300; KAT3B; RSTS2
NCBI Protein Information:	histone acetyltransferase p300
UniProt Protein Name:	Histone acetyltransferase p300
UniProt Synonym Protein Names:	E1A-associated protein p300
Protein Family:	Histone acetyltransferase
UniProt Gene Name:	EP300
UniProt Entry Name:	EP300_HUMAN

Component	Quantity
96-Well Cell Culture Clear-Bottom Microplate	2 plates
10X TBS	24 mL
Quenching Buffer	24 mL
Blocking Buffer	50 mL
15X Wash Buffer	50 mL
Primary Antibody Diluent	12 mL
100x Anti-Phospho Target Antibody	60 µL
100x Anti-Target Antibody	60 µL
Anti-GAPDH Antibody	60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody	12 mL
HRP-Conjugated Anti-Mouse IgG Antibody	12 mL
SDS Solution	12 mL
Stop Solution	24 mL
Ready-to-Use Substrate	12 mL
Crystal Violet Solution	12 mL
Adhesive Plate Seals	2 seals

The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:

Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
Micropipettes with capability of measuring volumes ranging from 1 µL to 1 ml
37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
Graph paper or computer software capable of generating or displaying logarithmic functions
Absorbent papers or vacuum aspirator
Test tubes or microfuge tubes capable of storing ≥1 ml
Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
Orbital shaker (optional)
Deionized or sterile water

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Step	Procedure
1.	Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µL of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37°C prior to adding cells.
2.	Incubate the cells for overnight at 37°C, 5% CO2.
3.	Treat the cells as desired.
4.	Remove the cell culture medium and rinse with 200 µL of 1x TBS, twice.
5.	Fix the cells by incubating with 100 µL of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells.
6.	Remove the Fixing Solution and wash the plate 3 times with 200 µL 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4°C for a week.
7.	Add 100 µL of Quenching Buffer and incubate for 20 minutes at room temperature.
8.	Wash the plate 3 times with 1x Wash Buffer for 5 minutes each time.
9.	Add 200 µL of Blocking Buffer and incubate for 1 hour at room temperature.
10.	Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
11.	Add 50 µL of 1x primary antibodies (Anti-p300 Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4°C. If the target expression is known to be high, incubate for 2 hours at room temperature.
12.	Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
13.	Add 50 µL of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature.
14.	Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
15.	Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark.
16.	Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader.

(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)