Histone H2A.X (Phospho-Ser139)Colorimetric Cell-Based ELISA Kit (CBCAB00071)

Product Name:	Histone H2A.X (Phospho-Ser139)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00071
ELISA Type:	Cell-Based
Target:	Histone H2A.X (Phospho-Ser139)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The Histone H2A.X (Phospho-Ser139) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Histone H2A.X protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Histone H2A.X in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Histone H2A.X phosphorylation.

Qualitative determination of Histone H2A.X (Phospho-Ser139) concentration is achieved by an indirect ELISA format. In essence, Histone H2A.X (Phospho-Ser139) is captured by Histone H2A.X (Phospho-Ser139)-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: 3014, UniProt ID: P16104, OMIM: 601772, Unigene: Hs.477879
Gene Symbol:	H2AFX
Sub Type:	Phospho

UniProt Protein Function:	H2AX: a histone that replaces conventional H2A in a subset of nucleosomes. Required for checkpoint-mediated arrest of cell cycle progression in response to low doses of ionizing radiation and for efficient repair of DNA double strand breaks (DSBs) specifically when modified by C-terminal phosphorylation. Histones are basic nuclear proteins that are responsible for the nucleosome structure of the chromosomal fiber in eukaryotes. Two molecules of each of the four core histones (H2A, H2B, H3, and H4) form an octamer, around which approximately 146 bp of DNA is wrapped in repeating units, called nucleosomes. Nucleosomes wrap and compact DNA into chromatin, limiting DNA accessibility to the cellular machineries which require DNA as a template. Histones thereby play a central role in transcription regulation, DNA repair, DNA replication and chromosomal stability. DNA accessibility is regulated via a complex set of post-translational modifications of histones, also called histone code, and nucleosome remodeling. Phosphorylated on S139 by ATM and DNA-PK in response to DNA double strand breaks (DSBs) generated by exogenous genotoxic agents and by stalled replication forks, and may also occur during meiotic recombination events and immunoglobulin class switching in lymphocytes. Phosphorylation can extend up to several thousand nucleosomes from the actual site of the DSB and may mark the surrounding chromatin for recruitment of proteins required for DNA damage signaling and repair. Widespread phosphorylation may also serve to amplify the damage signal or aid repair of persistent lesions. Dephosphorylation of S139 by PP2A is required for DNA DSB repair. Apparently phosphorylated on Y143 by WSTF, determining the relative recruitment of either DNA repair or pro-apoptotic factors. H2AXpY142 favors the recruitment of pro-apoptotic factors APBB1 and JNK1. In contrast, dephosphorylation of pY143 by EYA phosphatases favors the recruitment of MDC1-containing DNA repair complexes to the tail of phosphorylated pS139. Monoubiquitination of K119 by RING1 and RNF2/RING2 complex gives a specific tag for epigenetic transcriptional repression. Following DNA double-strand breaks (DSBs), it is ubiquitinated through 'K63' linkages by the E2 ligase UBE2N and the E3 ligases RNF8 and RNF168, leading to the recruitment of repair proteins to sites of DNA damage.
UniProt Protein Details:	Protein type:DNA repair, damage; DNA-binding; Helicase Chromosomal Location of Human Ortholog: 11q23.3 Cellular Component: chromosome, telomeric region; condensed nuclear chromosome; male germ cell nucleus; nuclear chromatin; nucleoplasm; nucleosome; nucleus; replication fork; XY body Molecular Function:damaged DNA binding; DNA binding; enzyme binding; histone binding; protein binding; protein heterodimerization activity Biological Process: cerebral cortex development; chromatin silencing; DNA damage checkpoint; DNA repair; double-strand break repair; double-strand break repair via homologous recombination; double-strand break repair via nonhomologous end joining; meiotic cell cycle; nucleosome assembly; positive regulation of DNA repair; response to DNA damage stimulus; response to ionizing radiation; spermatogenesis; viral reproduction
NCBI Summary:	Histones are basic nuclear proteins that are responsible for the nucleosome structure of the chromosomal fiber in eukaryotes. Two molecules of each of the four core histones (H2A, H2B, H3, and H4) form an octamer, around which approximately 146 bp of DNA is wrapped in repeating units, called nucleosomes. The linker histone, H1, interacts with linker DNA between nucleosomes and functions in the compaction of chromatin into higher order structures. This gene encodes a replication-independent histone that is a member of the histone H2A family, and generates two transcripts through the use of the conserved stem-loop termination motif, and the polyA addition motif. [provided by RefSeq, Oct 2015]
UniProt Code:	P16104
NCBI GenInfo Identifier:	121992
NCBI Gene ID:	3014
NCBI Accession:	P16104.2
UniProt Secondary Accession:	P16104,Q4ZGJ7, Q6IAS5,
UniProt Related Accession:	P16104
Molecular Weight:	15,145 Da
NCBI Full Name:	Histone H2AX
NCBI Synonym Full Names:	H2A histone family member X
NCBI Official Symbol:	H2AFX
NCBI Official Synonym Symbols:	H2AX; H2A.X; H2A/X
NCBI Protein Information:	histone H2AX
UniProt Protein Name:	Histone H2AX
UniProt Synonym Protein Names:	Histone H2A.X
UniProt Gene Name:	H2AFX
UniProt Entry Name:	H2AX_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-Histone H2A.X (Phospho-Ser139) Antibody, Anti-Histone H2A.X 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.)