MRE11 Colorimetric Cell-Based ELISA Kit (CBCAB00083)

Product Name:	MRE11 Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00083
ELISA Type:	Cell-Based
Target:	MRE11
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

The MRE11 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect MRE11 protein expression profile in cells. The kit can be used for measuring the relative amounts of MRE11 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on MRE11.

Qualitative determination of MRE11 concentration is achieved by an indirect ELISA format. In essence, MRE11 is captured by MRE11-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: 4361, UniProt ID: P49959, OMIM: 600814/604391, Unigene: Hs.192649
Gene Symbol:	MRE11A
Sub Type:	None

UniProt Protein Function:	MRE11A: a nuclear protein involved in homologous recombination, telomere length maintenance, and DNA double-strand break repair. By itself, the protein has 3' to 5' exonuclease activity and endonuclease activity. The protein forms a complex with the RAD50 homolog; this complex is required for nonhomologous joining of DNA ends and possesses increased single-stranded DNA endonuclease and 3' to 5' exonuclease activities. In conjunction with a DNA ligase, this protein promotes the joining of noncomplementary ends in vitro using short homologies near the ends of the DNA fragments. Alternative splicing results in two different isoforms.
UniProt Protein Details:	Protein type:DNA-binding; Cell cycle regulation; DNA repair, damage; Deoxyribonuclease Chromosomal Location of Human Ortholog: 11q21 Cellular Component: nucleoplasm; chromosome, telomeric region; Mre11 complex; cytosol; nucleus; chromatin Molecular Function:protein C-terminus binding; ATP-dependent DNA helicase activity; protein binding; nuclease activity; DNA binding; single-stranded DNA specific endodeoxyribonuclease activity; manganese ion binding; endonuclease activity; double-stranded DNA binding; 3'-5' exonuclease activity; endodeoxyribonuclease activity Biological Process: positive regulation of kinase activity; sister chromatid cohesion; negative regulation of DNA endoreduplication; telomere maintenance via telomerase; DNA repair; regulation of mitotic recombination; DNA catabolic process, endonucleolytic; positive regulation of protein amino acid autophosphorylation; DNA duplex unwinding; double-strand break repair via nonhomologous end joining; double-strand break repair via homologous recombination; DNA recombination; cell proliferation; intra-S DNA damage checkpoint; base-excision repair; nucleotide-excision repair; meiotic recombination; double-strand break repair; mitotic cell cycle G2/M transition DNA damage checkpoint; synapsis; positive regulation of interferon type I production; innate immune response; telomere maintenance; response to DNA damage stimulus Disease: Ataxia-telangiectasia-like Disorder 1
NCBI Summary:	This gene encodes a nuclear protein involved in homologous recombination, telomere length maintenance, and DNA double-strand break repair. By itself, the protein has 3' to 5' exonuclease activity and endonuclease activity. The protein forms a complex with the RAD50 homolog; this complex is required for nonhomologous joining of DNA ends and possesses increased single-stranded DNA endonuclease and 3' to 5' exonuclease activities. In conjunction with a DNA ligase, this protein promotes the joining of noncomplementary ends in vitro using short homologies near the ends of the DNA fragments. This gene has a pseudogene on chromosome 3. Alternative splicing of this gene results in two transcript variants encoding different isoforms. [provided by RefSeq, Jul 2008]
UniProt Code:	P49959
NCBI GenInfo Identifier:	17380137
NCBI Gene ID:	4361
NCBI Accession:	P49959.3
UniProt Secondary Accession:	P49959,O43475, B3KTC7,
UniProt Related Accession:	P49959
Molecular Weight:	708
NCBI Full Name:	Double-strand break repair protein MRE11A
NCBI Synonym Full Names:	MRE11 meiotic recombination 11 homolog A (S. cerevisiae)
NCBI Official Symbol:	MRE11A
NCBI Official Synonym Symbols:	ATLD; HNGS1; MRE11; MRE11B
NCBI Protein Information:	double-strand break repair protein MRE11A; AT-like disease; MRE11 homolog 1; MRE11 homolog A; endo/exonuclease Mre11; meiotic recombination 11 homolog 1; meiotic recombination 11 homolog A; DNA recombination and repair protein
UniProt Protein Name:	Double-strand break repair protein MRE11A
UniProt Synonym Protein Names:	Meiotic recombination 11 homolog 1; MRE11 homolog 1; Meiotic recombination 11 homolog A; MRE11 homolog A
Protein Family:	Double-strand break repair protein
UniProt Gene Name:	MRE11A
UniProt Entry Name:	MRE11_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-MRE11 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.)