Ku70/XRCC6 Colorimetric Cell-Based ELISA Kit (CBCAB00735)

Product Name:	Ku70/XRCC6 Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00735
ELISA Type:	Cell-Based
Target:	Ku70/XRCC6
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

The Ku70/XRCC6 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Ku70/XRCC6 protein expression profile in cells. The kit can be used for measuring the relative amounts of Ku70/XRCC6 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on Ku70/XRCC6.

Qualitative determination of Ku70/XRCC6 concentration is achieved by an indirect ELISA format. In essence, Ku70/XRCC6 is captured by Ku70/XRCC6-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: 2547, UniProt ID: P12956, OMIM: 152690, Unigene: Hs.292493
Gene Symbol:	XRCC6
Sub Type:	None

UniProt Protein Function:	Ku70: a mini-chromosome maintenance protein, essential for the initiation of eukaryotic genome replication. Allows DNA to undergo a single round of replication per cell cycle. Required for the entry in S phase and for cell division.
UniProt Protein Details:	Protein type:Helicase; DNA repair, damage; EC 3.6.4.-; Nuclear receptor co-regulator; DNA-binding; RNA-binding; EC 4.2.99.- Chromosomal Location of Human Ortholog: 22q13.2 Cellular Component: cytosol; membrane; nuclear chromosome, telomeric region; nuclear telomere cap complex; nucleoplasm; nucleus; transcription factor complex Molecular Function:5'-deoxyribose-5-phosphate lyase activity; damaged DNA binding; double-stranded DNA binding; double-stranded telomeric DNA binding; protein binding; protein C-terminus binding Biological Process: DNA ligation; DNA recombination; double-strand break repair via nonhomologous end joining; negative regulation of transcription, DNA-dependent; positive regulation of interferon type I production; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription, DNA-dependent; protein heterotetramerization; regulation of smooth muscle cell proliferation; telomere maintenance
NCBI Summary:	The p70/p80 autoantigen is a nuclear complex consisting of two subunits with molecular masses of approximately 70 and 80 kDa. The complex functions as a single-stranded DNA-dependent ATP-dependent helicase. The complex may be involved in the repair of nonhomologous DNA ends such as that required for double-strand break repair, transposition, and V(D)J recombination. High levels of autoantibodies to p70 and p80 have been found in some patients with systemic lupus erythematosus. [provided by RefSeq, Jul 2008]
UniProt Code:	P12956
NCBI GenInfo Identifier:	125729
NCBI Gene ID:	2547
NCBI Accession:	P12956.2
UniProt Secondary Accession:	P12956,Q6FG89, Q9UCQ2, Q9UCQ3, B1AHC8,
UniProt Related Accession:	P12956
Molecular Weight:	65,149 Da
NCBI Full Name:	X-ray repair cross-complementing protein 6
NCBI Synonym Full Names:	X-ray repair cross complementing 6
NCBI Official Symbol:	XRCC6
NCBI Official Synonym Symbols:	ML8; KU70; TLAA; CTC75; CTCBF; G22P1
NCBI Protein Information:	X-ray repair cross-complementing protein 6
UniProt Protein Name:	X-ray repair cross-complementing protein 6
UniProt Synonym Protein Names:	5'-deoxyribose-5-phosphate lyase Ku70; 5'-dRP lyase Ku70; 70 kDa subunit of Ku antigen; ATP-dependent DNA helicase 2 subunit 1; ATP-dependent DNA helicase II 70 kDa subunit; CTC box-binding factor 75 kDa subunit; CTC75; CTCBF; DNA repair protein XRCC6; Lupus Ku autoantigen protein p70; Ku70; Thyroid-lupus autoantigen; TLAA; X-ray repair complementing defective repair in Chinese hamster cells 6
Protein Family:	X-ray repair cross-complementing protein
UniProt Gene Name:	XRCC6
UniProt Entry Name:	XRCC6_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-Ku70/XRCC6 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.)