Chk2 (Phospho-Thr383) Colorimetric Cell-Based ELISA Kit

Product Name:	Chk2 (Phospho-Thr383) Colorimetric Cell-Based ELISA
Product Code:	CBCAB01370
ELISA Type:	Cell-Based
Target:	Chk2 (Phospho-Thr383)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The Chk2 (Phospho-Thr383) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Chk2 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Chk2 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Chk2 phosphorylation.

Qualitative determination of Chk2 (Phospho-Thr383) concentration is achieved by an indirect ELISA format. In essence, Chk2 (Phospho-Thr383) is captured by Chk2 (Phospho-Thr383)-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: 11200, UniProt ID: O96017, OMIM: 176807/259500/604373/609265, Unigene: Hs.291363/Hs.505297
Gene Symbol:	CHEK2
Sub Type:	Phospho

UniProt Protein Function:	Chk2: a CAMK protein kinase of the Rad53 family. A cell cycle checkpoint regulator and putative tumor suppressor. It contains a forkhead-associated protein interaction domain essential for activation in response to DNA damage and is rapidly phosphorylated in response to replication blocks and DNA damage. Phosphorylates and inhibits CDC25C, preventing entry into mitosis, p53, leading to cell cycle arrest in G1, and BRCA1, restoring survival after DNA damage. Twelve splice variant isoforms have been described for human Chk2. LOF mutants cause Li-Fraumeni syndrome, a highly penetrant familial cancer phenotype also caused by p53 mutations. Familial mutations also associated with prostate and breast cancer, and mutations also seen in a variety of sporadic cancers and cell lines.
UniProt Protein Details:	Protein type:EC 2.7.11.1; Kinase, protein; Protein kinase, Ser/Thr (non-receptor); Protein kinase, CAMK; Tumor suppressor; CAMK group; RAD53 family Chromosomal Location of Human Ortholog: 22q12.1 Cellular Component: nucleoplasm; Golgi apparatus; PML body; chromosome, telomeric region Molecular Function:protein serine/threonine kinase activity; identical protein binding; protein binding; protein homodimerization activity; metal ion binding; ubiquitin protein ligase binding; protein kinase binding; ATP binding Biological Process: DNA damage induced protein phosphorylation; transcription, DNA-dependent; protein stabilization; protein amino acid autophosphorylation; positive regulation of transcription, DNA-dependent; DNA damage response, signal transduction; protein amino acid phosphorylation; regulation of transcription, DNA-dependent; DNA damage response, signal transduction resulting in induction of apoptosis; cell division; cellular protein catabolic process; double-strand break repair; DNA damage checkpoint; response to gamma radiation; regulation of protein catabolic process; G2/M transition of mitotic cell cycle; response to DNA damage stimulus Disease: Li-fraumeni Syndrome 2; Breast Cancer; Prostate Cancer; Osteogenic Sarcoma
NCBI Summary:	In response to DNA damage and replication blocks, cell cycle progression is halted through the control of critical cell cycle regulators. The protein encoded by this gene is a cell cycle checkpoint regulator and putative tumor suppressor. It contains a forkhead-associated protein interaction domain essential for activation in response to DNA damage and is rapidly phosphorylated in response to replication blocks and DNA damage. When activated, the encoded protein is known to inhibit CDC25C phosphatase, preventing entry into mitosis, and has been shown to stabilize the tumor suppressor protein p53, leading to cell cycle arrest in G1. In addition, this protein interacts with and phosphorylates BRCA1, allowing BRCA1 to restore survival after DNA damage. Mutations in this gene have been linked with Li-Fraumeni syndrome, a highly penetrant familial cancer phenotype usually associated with inherited mutations in TP53. Also, mutations in this gene are thought to confer a predisposition to sarcomas, breast cancer, and brain tumors. This nuclear protein is a member of the CDS1 subfamily of serine/threonine protein kinases. Several transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Apr 2012]
UniProt Code:	O96017
NCBI GenInfo Identifier:	6685284
NCBI Gene ID:	11200
NCBI Accession:	O96017.1
UniProt Secondary Accession:	O96017,Q6QA03, Q6QA04, Q6QA05, Q6QA06, Q6QA07, Q6QA08 Q6QA10, A8K3Y9, B7ZBF3, B7ZBF4, B7ZBF5,
UniProt Related Accession:	O96017
Molecular Weight:	543
NCBI Full Name:	Serine/threonine-protein kinase Chk2
NCBI Synonym Full Names:	checkpoint kinase 2
NCBI Official Symbol:	CHEK2
NCBI Official Synonym Symbols:	CDS1; CHK2; LFS2; RAD53; hCds1; HuCds1; PP1425
NCBI Protein Information:	serine/threonine-protein kinase Chk2; cds1 homolog; CHK2 checkpoint homolog; checkpoint-like protein CHK2
UniProt Protein Name:	Serine/threonine-protein kinase Chk2
UniProt Synonym Protein Names:	CHK2 checkpoint homolog; Cds1 homolog; Hucds1; hCds1; Checkpoint kinase 2
Protein Family:	Serine/threonine-protein kinase
UniProt Gene Name:	CHEK2
UniProt Entry Name:	CHK2_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-Chk2 (Phospho-Thr383) Antibody, Anti-Chk2 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.)

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Chk2 (Phospho-Thr383) Colorimetric Cell-Based ELISA Kit

Description