ATF2 (Phospho-Thr71)Colorimetric Cell-Based ELISA Kit (CBCAB01494)

Product Name:	ATF2 (Phospho-Thr71)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB01494
ELISA Type:	Cell-Based
Target:	ATF2 (Phospho-Thr71)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The ATF2 (Phospho-Thr71) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect ATF2 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated ATF2 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on ATF2 phosphorylation.

Qualitative determination of ATF2 (Phospho-Thr71) concentration is achieved by an indirect ELISA format. In essence, ATF2 (Phospho-Thr71) is captured by ATF2 (Phospho-Thr71)-specific primary (1Ãƒ¸) antibodies while the HRP-conjugated secondary (2Ãƒ¸) antibodies bind the Fc region of the 1Ãƒ¸ antibody. Through this binding, the HRP enzyme conjugated to the 2Ãƒ¸ 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: 1386, UniProt ID: P15336, OMIM: 123811, Unigene: Hs.592510
Gene Symbol:	ATF2
Sub Type:	Phospho

UniProt Protein Function:	ATF-2: a transcription factor that is a member of the leucine zipper family. Binds to the cAMP-responsive element (CRE). Forms a homodimer or heterodimer with c-Jun and stimulates CRE-dependent transcription. Also possesses histone acetyltransferase (HAT) activity that specifically acetylates histones H2B and H4 in vitro.
UniProt Protein Details:	Protein type:EC 2.3.1.48; Transcription factor; C2H2-type zinc finger protein Chromosomal Location of Human Ortholog: 2q32 Cellular Component: nucleoplasm; mitochondrial outer membrane; cytoplasm; nucleus Molecular Function:RNA polymerase II transcription factor activity, enhancer binding; protein binding; histone acetyltransferase activity; protein heterodimerization activity; metal ion binding; cAMP response element binding protein binding; transcription coactivator activity; chromatin binding; transcription factor activity; protein kinase binding Biological Process: fat cell differentiation; transcription from RNA polymerase II promoter; establishment and/or maintenance of chromatin architecture; MyD88-independent toll-like receptor signaling pathway; stress-activated MAPK cascade; toll-like receptor 3 signaling pathway; positive regulation of transforming growth factor-beta2 production; response to osmotic stress; toll-like receptor 2 signaling pathway; toll-like receptor 10 signaling pathway; MyD88-dependent toll-like receptor signaling pathway; toll-like receptor 5 signaling pathway; regulation of transcription from RNA polymerase II promoter; regulation of transcription, DNA-dependent; regulation of transcription factor activity; intra-S DNA damage checkpoint; toll-like receptor signaling pathway; innate immune response; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription factor activity; histone acetylation; toll-like receptor 9 signaling pathway; response to DNA damage stimulus; toll-like receptor 4 signaling pathway
NCBI Summary:	This gene encodes a transcription factor that is a member of the leucine zipper family of DNA binding proteins. The encoded protein has been identified as a moonlighting protein based on its ability to perform mechanistically distinct functions This protein binds to the cAMP-responsive element (CRE), an octameric palindrome. It forms a homodimer or a heterodimer with c-Jun and stimulates CRE-dependent transcription. This protein is also a histone acetyltransferase (HAT) that specifically acetylates histones H2B and H4 in vitro; thus it may represent a class of sequence-specific factors that activate transcription by direct effects on chromatin components. The encoded protein may also be involved in cell's DNA damage response independent of its role in transcriptional regulation. Several alternatively spliced transcript variants have been found for this gene [provided by RefSeq, Jan 2014]
UniProt Code:	P15336
NCBI GenInfo Identifier:	215274241
NCBI Gene ID:	1386
NCBI Accession:	P15336.4
UniProt Secondary Accession:	P15336,Q13000, Q3B7B7, Q4ZFU9, Q53RY2, Q8TAR1, A1L3Z2 A4D7U4, A4D7U5, A4D7V1, D3DPE9, G8JLM5,
UniProt Related Accession:	P15336
Molecular Weight:	505
NCBI Full Name:	Cyclic AMP-dependent transcription factor ATF-2
NCBI Synonym Full Names:	activating transcription factor 2
NCBI Official Symbol:	ATF2
NCBI Official Synonym Symbols:	HB16; CREB2; TREB7; CREB-2; CRE-BP1
NCBI Protein Information:	cyclic AMP-dependent transcription factor ATF-2; histone acetyltransferase ATF2; cAMP-dependent transcription factor ATF-2; cAMP-responsive element-binding protein 2; cAMP response element-binding protein CRE-BP1; cyclic AMP-responsive element-binding protein 2; cAMP responsive element binding protein 2, formerly; activating transcription factor 2 splice variant ATF2-var2
UniProt Protein Name:	Cyclic AMP-dependent transcription factor ATF-2
UniProt Synonym Protein Names:	Activating transcription factor 2; Cyclic AMP-responsive element-binding protein 2; CREB-2; cAMP-responsive element-binding protein 2; HB16; Histone acetyltransferase ATF2; cAMP response element-binding protein CRE-BP1
Protein Family:	Alcohol O-acetyltransferase
UniProt Gene Name:	ATF2
UniProt Entry Name:	ATF2_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-ATF2 (Phospho-Thr71) Antibody, Anti-ATF2 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.)