RAD17 (Phospho-Ser645)Colorimetric Cell-Based ELISA Kit (CBCAB01250)

Product Name:	RAD17 (Phospho-Ser645)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB01250
ELISA Type:	Cell-Based
Target:	RAD17 (Phospho-Ser645)
Reactivity:	Human, Mouse
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The RAD17 (Phospho-Ser645) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect RAD17 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated RAD17 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on RAD17 phosphorylation.

Qualitative determination of RAD17 (Phospho-Ser645) concentration is achieved by an indirect ELISA format. In essence, RAD17 (Phospho-Ser645) is captured by RAD17 (Phospho-Ser645)-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: 5884, UniProt ID: O75943, OMIM: 603139, Unigene: Hs.16184
Gene Symbol:	RAD17
Sub Type:	Phospho

UniProt Protein Function:	RAD17: a cell cycle checkpoint gene required for cell cycle arrest and DNA damage repair in response to DNA damage. Shares strong similarity with DNA replication factor C (RFC), and can form a complex with RFCs. Binds to chromatin prior to DNA damage and is phosphorylated by ATR after the damage. Phosphorylated and activated after DNA damage, inducing cell cycle G2 arrest. Eight alternatively spliced variants have been reported.
UniProt Protein Details:	Protein type:Cell cycle regulation Chromosomal Location of Human Ortholog: 5q13 Cellular Component: nucleoplasm; chromosome, telomeric region; nucleus Molecular Function:protein binding; ATP binding Biological Process: regulation of phosphorylation; negative regulation of DNA replication; mitotic cell cycle checkpoint; DNA damage checkpoint; DNA repair; DNA replication; DNA replication checkpoint; response to DNA damage stimulus
NCBI Summary:	The protein encoded by this gene is highly similar to the gene product of Schizosaccharomyces pombe rad17, a cell cycle checkpoint gene required for cell cycle arrest and DNA damage repair in response to DNA damage. This protein shares strong similarity with DNA replication factor C (RFC), and can form a complex with RFCs. This protein binds to chromatin prior to DNA damage and is phosphorylated by the checkpoint kinase ATR following damage. This protein recruits the RAD1-RAD9-HUS1 checkpoint protein complex onto chromatin after DNA damage, which may be required for its phosphorylation. The phosphorylation of this protein is required for the DNA-damage-induced cell cycle G2 arrest, and is thought to be a critical early event during checkpoint signaling in DNA-damaged cells. Multiple alternatively spliced transcript variants of this gene, which encode four distinct protein isoforms, have been reported. Two pseudogenes, located on chromosomes 7 and 13, have been identified. [provided by RefSeq, Jul 2013]
UniProt Code:	O75943
NCBI GenInfo Identifier:	62287484
NCBI Gene ID:	5884
NCBI Accession:	O75943.2
UniProt Secondary Accession:	O75943,O75714, Q7Z3S4, Q9UNK7, Q9UNR7, Q9UNR8, Q9UPF5 A8K8X2, D3DWA5,
UniProt Related Accession:	O75943
Molecular Weight:	681
NCBI Full Name:	Cell cycle checkpoint protein RAD17
NCBI Synonym Full Names:	RAD17 homolog (S. pombe)
NCBI Official Symbol:	RAD17
NCBI Official Synonym Symbols:	CCYC; R24L; RAD24; HRAD17; RAD17SP
NCBI Protein Information:	cell cycle checkpoint protein RAD17; RAD1 homolog; Rad17-like protein; RF-C activator 1 homolog; RF-C/activator 1 homolog
UniProt Protein Name:	Cell cycle checkpoint protein RAD17
UniProt Synonym Protein Names:	RF-C/activator 1 homolog
Protein Family:	Checkpoint protein
UniProt Gene Name:	RAD17
UniProt Entry Name:	RAD17_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-RAD17 (Phospho-Ser645) Antibody, Anti-RAD17 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.)