CREB (Phospho-Ser133)Colorimetric Cell-Based ELISA Kit (CBCAB00050)

Product Name:	CREB (Phospho-Ser133)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00050
ELISA Type:	Cell-Based
Target:	CREB (Phospho-Ser133)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The CREB (Phospho-Ser133) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect CREB protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated CREB in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on CREB phosphorylation.

Qualitative determination of CREB (Phospho-Ser133) concentration is achieved by an indirect ELISA format. In essence, CREB (Phospho-Ser133) is captured by CREB (Phospho-Ser133)-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: 1385, UniProt ID: P16220, OMIM: 123810/612160, Unigene: Hs.516646/Hs.584750
Gene Symbol:	CREB1
Sub Type:	Phospho

UniProt Protein Function:	Function: This protein binds the cAMP response element (CRE), a sequence present in many viral and cellular promoters. CREB stimulates transcription on binding to the CRE. Transcription activation is enhanced by the TORC coactivators which act independently of Ser-133 phosphorylation. Implicated in synchronization of circadian rhythmicity.
UniProt Protein Details:	Subunit structure: Interacts with PPRC1. Binds DNA as a dimer. This dimer is stabilized by magnesium ions. Interacts, through the bZIP domain, with the coactivators TORC1/CRTC1, TORC2/CRTC2 and TORC3/CRTC3. When phosphorylated on Ser-133, binds CREBBP By similarity. Interacts (phosporylated form) with TOX3. Interacts with ARRB1. Ref.8 Ref.9 Ref.11 Ref.13 Ref.14 Ref.15 Ref.16 Ref.20 Subcellular location: Nucleus Ref.12. Post-translational modification: Stimulated by phosphorylation. Phosphorylation of both Ser-133 and Ser-142 in the SCN regulates the activity of CREB and participates in circadian rhythm generation. Phosphorylation of Ser-133 allows CREBBP binding By similarity. Phosphorylated upon DNA damage, probably by ATM or ATR. Ref.10 Ref.17 Ref.19Sumoylated by SUMO1. Sumoylation on Lys-304, but not on Lys-285, is required for nuclear localization of this protein. Sumoylation is enhanced under hypoxia, promoting nuclear localization and stabilization. Ref.12 Involvement in Disease: Defects in CREB1 may be a cause of angiomatoid fibrous histiocytoma (AFH) [ MIM:612160]. A distinct variant of malignant fibrous histiocytoma that typically occurs in children and adolescents and is manifest by nodular subcutaneous growth. Characteristic microscopic features include lobulated sheets of histiocyte-like cells intimately associated with areas of hemorrhage and cystic pseudovascular spaces, as well as a striking cuffing of inflammatory cells, mimicking a lymph node metastasis. Note=A chromosomal aberration involving CREB1 is found in a patient with angiomatoid fibrous histiocytoma. Translocation t(2;22)(q33;q12) with CREB1 generates a EWSR1/CREB1 fusion gene that is most common genetic abnormality in this tumor type. Sequence similarities: Belongs to the bZIP family.Contains 1 bZIP domain.Contains 1 KID (kinase-inducible) domain.
NCBI Summary:	This gene encodes a transcription factor that is a member of the leucine zipper family of DNA binding proteins. This protein binds as a homodimer to the cAMP-responsive element, an octameric palindrome. The protein is phosphorylated by several protein kinases, and induces transcription of genes in response to hormonal stimulation of the cAMP pathway. Alternate splicing of this gene results in two transcript variants encoding different isoforms. [provided by RefSeq]
UniProt Code:	P16220
NCBI GenInfo Identifier:	117434
NCBI Gene ID:	1385
NCBI Accession:	P16220.2
UniProt Secondary Accession:	P16220,P21934, Q9UMA7,
UniProt Related Accession:	P16220,Q16366,Q16367,Q4ZG78,Q53RU9,Q53X93,Q5U0J5,Q6V963
Molecular Weight:	40 kDa
NCBI Full Name:	Cyclic AMP-responsive element-binding protein 1
NCBI Synonym Full Names:	cAMP responsive element binding protein 1
NCBI Official Symbol:	CREB1
NCBI Official Synonym Symbols:	CREB; MGC9284
NCBI Protein Information:	cyclic AMP-responsive element-binding protein 1; CREB-1; OTTHUMP00000163864; OTTHUMP00000163865; OTTHUMP00000206660; OTTHUMP00000206662; OTTHUMP00000206667; transactivator protein; active transcription factor CREB; cAMP-response element-binding protein-1; cAMP-responsive element-binding protein 1
UniProt Protein Name:	Cyclic AMP-responsive element-binding protein 1
Protein Family:	CREB-binding protein
UniProt Gene Name:	CREB1
UniProt Entry Name:	CREB1_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 (Phospho-Ser133) Antibody, Anti-CREB 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.)