CREB-BP Colorimetric Cell-Based ELISA Kit (CBCAB00599)

Product Name:	CREB-BP Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00599
ELISA Type:	Cell-Based
Target:	CREB-BP
Reactivity:	Human, Mouse
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

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

Qualitative determination of CREB-BP concentration is achieved by an indirect ELISA format. In essence, CREB-BP is captured by CREB-BP-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: 1387, UniProt ID: Q92793, OMIM: 600140, Unigene: Hs.459759
Gene Symbol:	CREBBP
Sub Type:	None

UniProt Protein Function:	CBP: a protein acetyltransferase that can transcriptionally activate histones. Acetylates the NCOA3 coactivator. Binds specifically to phosphorylated CREB1 and enhances its transcriptional activity toward cAMP-responsive genes. Methylation of the KIX domain by CARM1 blocks association with CREB, blocking CREB signaling, and activating the apoptotic response. Found in a complex containing NCOA2, NCOA3, IKKA, IKKB, and IKBKG. Probably part of a complex with HIF1A and EP300. Interacts with the C-terminal region of CITED4. The TAZ-type 1 domain interacts with HIF1A. Interacts with MAF, SRCAP, CARM1, ELF3, MLLT7/FOXO4, N4BP2, NCOA1, NCOA3, NCOA6, PCAF, PELP1, PML, SMAD1, SMAD2, SMAD3, SPIB and TRERF1. Interacts with HTLV-1 Tax, p30II, and HIV-1 Tat. Interacts with KLF1; the interaction results in acetylation of KLF1 and enhancement of its transcriptional activity. Interacts with ZCCHC12. Interacts with DAXX; the interaction is dependent on CBP sumoylation and results in suppression of the transcriptional activiy via recruitment of HDAC2 to DAAX. Interacts with MTDH. Interacts with NFATC4. Interacts with MAFG; the interaction acetylates MAFG in the basic region and stimulates NFE2 transcriptional activity through increasing its DNA-binding activity. Interacts with IRF2; the interaction acetylates IRF2 and regulates its activity on the H4 promoter. Interacts via its N-terminus with the C-terminus of SS18L1. Interacts with MECOM. Chromosomal aberrations involving CBP may be a cause of acute myeloid leukemias. Known translocation partners include MYST3, MLL, and MYST4. MYST3-CBP fusion proteins may induce leukemia by inhibiting RUNX1-mediated transcription. Defects in CBP are a cause of Rubinstein-Taybi syndrome type 1 (RSTS1), an autosomal dominant disorder characterized by craniofacial abnormalities, broad thumbs, broad big toes, mental retardation and a propensity for development of malignancies.
UniProt Protein Details:	Protein type:Nuclear receptor co-regulator; EC 2.3.1.48; DNA-binding; Acetyltransferase; Motility/polarity/chemotaxis; Transcription, coactivator/corepressor Chromosomal Location of Human Ortholog: 16p13.3 Cellular Component: nucleoplasm; nuclear body; transcription factor complex; nuclear chromatin; cytoplasm; outer kinetochore of condensed chromosome; nucleus; histone acetyltransferase complex Molecular Function:MRF binding; signal transducer activity; protein binding; histone acetyltransferase activity; zinc ion binding; p53 binding; acetyltransferase activity; transcription coactivator activity; chromatin binding; transcription factor activity; transcription factor binding Biological Process: transcription initiation from RNA polymerase II promoter; establishment and/or maintenance of chromatin architecture; Notch signaling pathway; viral reproduction; positive regulation of transcription, DNA-dependent; rhythmic process; germ-line stem cell maintenance; negative regulation of transcription from RNA polymerase II promoter; cellular lipid metabolic process; signal transduction; homeostatic process; regulation of transcription, DNA-dependent; response to hypoxia; positive regulation of interferon type I production; innate immune response; positive regulation of transcription from RNA polymerase II promoter; gene expression; protein complex assembly; embryonic digit morphogenesis; histone acetylation; regulation of smoothened signaling pathway; N-terminal peptidyl-lysine acetylation Disease: Rubinstein-taybi Syndrome 1
NCBI Summary:	This gene is ubiquitously expressed and is involved in the transcriptional coactivation of many different transcription factors. First isolated as a nuclear protein that binds to cAMP-response element binding protein (CREB), this gene is now known to play critical roles in embryonic development, growth control, and homeostasis by coupling chromatin remodeling to transcription factor recognition. The protein encoded by this gene has intrinsic histone acetyltransferase activity and also acts as a scaffold to stabilize additional protein interactions with the transcription complex. This protein acetylates both histone and non-histone proteins. This protein shares regions of very high sequence similarity with protein p300 in its bromodomain, cysteine-histidine-rich regions, and histone acetyltransferase domain. Mutations in this gene cause Rubinstein-Taybi syndrome (RTS). Chromosomal translocations involving this gene have been associated with acute myeloid leukemia. Alternative splicing results in multiple transcript variants encoding different isoforms. [provided by RefSeq, Feb 2009]
UniProt Code:	Q92793
NCBI GenInfo Identifier:	116241283
NCBI Gene ID:	1387
NCBI Accession:	Q92793.3
UniProt Secondary Accession:	Q92793,O00147, Q16376, Q4LE28, D3DUC9,
UniProt Related Accession:	Q92793
Molecular Weight:	260,993 Da
NCBI Full Name:	CREB-binding protein
NCBI Synonym Full Names:	CREB binding protein
NCBI Official Symbol:	CREBBP
NCBI Official Synonym Symbols:	CBP; RSTS; KAT3A
NCBI Protein Information:	CREB-binding protein
UniProt Protein Name:	CREB-binding protein
Protein Family:	CREB-binding protein
UniProt Gene Name:	CREBBP
UniProt Entry Name:	CBP_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-CREB-BP 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.)