OxiSelect™ Oxidative DNA Damage Quantitation Kit (AP Sites)

Catalog No. ABIN2344972

Product Details

Reactivity: Others

Detection Method: Colorimetric

Application: Quantification (Q)

Brand: OxiSelect™

Sample Type: Cell Samples, DNA samples, Tissue Samples

Sensitivity: Detect as few as 4-40 AP sites in 10⁵ bp DNA

Characteristics: The OxiSelect™ Oxidative DNA Damage Quantitation Kit (AP sites) uses an Aldehyde Reactive Probe (ARP) to react specifically with an aldehyde group on the open ring form of AP sites. This allows for the AP sites to be tagged with biotin which is later detected with Streptavidin-Enzyme conjugate. The quantity of AP sites in unknown DNA sample is determined by comparing its absorbance with a standard curve generated from the provided DNA standard containing predetermined AP sites. The kit has a detection sensitivity range of 4 to 40 AP sites per 1 x 105 bp. Each kit provides sufficient reagents to perform up to 10 assays, including standard curve and unknown samples.

Components:

1. Glycogen Solution : One 10 μL vial of 10 mg/mL glycogen.
2. Sodium Acetate Solution : One 200 μL of 3M Sodium Acetate, pH 5.5.
3. ARP Solution : One 50 μL vial of 10 mM ARP.
4. DNA High-Binding Plate : One strip well plate containing 32 wells (8 x 4). 2
5. DNA Binding Solution : One 1.25 mL vial.
6. 10X Wash Buffer : One 10 mL bottle.
7. Streptavidin-Enzyme Conjugate : One 10 μL vial.
8. Substrate Solution : One 4 mL amber bottle.
9. Stop Solution (Part. No. 310808-T): One 4 mL bottle.
10. Reduced DNA Standard : One 200 μL vial of 6 μg/mL fully reduced in TE Buffer (0 ARP/100,000 bp).
11. ARP-DNA Standard : One 75 μL vial of 6 μg/mL ARP-DNA in TE Buffer (40 ARP/100,000 bp).

Material not included:

1. DNA samples from cell or tissue for measuring DNA damage
2. TE Buffer: 10 mM Tris, pH 7.5, 1 mM EDTA
3. 100 % and 70 % Ethanol
4. 10 μL to 1000 μL adjustable single channel micropipettes with disposable tips
5. 50 μL to 300 μL adjustable multichannel micropipette with disposable tips
6. 37 °C Incubator
7. Multichannel micropipette reservoir
8. Microplate reader capable of reading at 450 nm (620 nm as optional reference wave length)

Application Details

Application Notes: Optimal working dilution should be determined by the investigator.

Comment:

• Detect as few as 4-40 AP sites in 10^5 bp DNA
• Suitable for use with cells or tissues
• Oxidized and reduced DNA standards included for absolute quantitation

Reagent Preparation:

• 1X Wash Buffer: Dilute the 10X Wash Buffer Concentrate to 1X with deionized water. Stir to homogeneity.
• Streptavidin-Enzyme Conjugate: Immediately before use, dilute the Streptavidin-Enzyme Conjugate 1:1000 with 1X Wash Buffer. Do not store diluted solutions.

Assay Procedure:

I. ARP Reaction

1. Isolate genomic DNA with desired method and dissolve the genomic DNA in TE buffer. Dilute the genomic DNA with TE buffer to 100 μg/mL. Note: During DNA extraction, avoid heating the DNA solution, or any procedure will introduce AP sites. We recommend using DNAZOL reagent to extract DNA and dissolve DNA in TE buffer.
2. Mix 5 μL of purified genomic DNA (100 μg/mL) with 5 μL o f ARP solution in a microcentrifuge tube and incubate 1 hr at 37 °C.
3. Add 90 μL of TE buffer and 1 μL of Glycogen Solution to each tube and mix well.
4. Add 10 μL of Sodium Acetate Solution to each tube, mix well.
5. Add 300 μL of absolute ethanol to each tube and mix well and incubate at -20 °C for 30 minutes.
6. Centrifuge for 10-20 minutes at 14,000 g and carefully wash the pellet three times with 70 % ethanol.
7. Dissolve the DNA pellet in 10-50 μL of TE buffer and determine the DNA concentration with desired method. ARP-derived DNA can be stored at -20 °C for up to one year. Note: It is important that the ARP-derived DNA concentration is determined precisely for the accurate measurement of AP sites. We recommend using Invitrogen's Quanti-iT™ DNA assay kit to measure DNA concentration. 4

II. Determination of AP sites in DNA:

1. Dilute the ARP-derived DNA sample to 1 μg/mL with TE buffer.
2. Add 50 μL of ARP-derived DNA sample or each dilution of the prepared ARP-DNA standards to the DNA High-binding plate. Add 50 μL of DNA Binding Solution to each well. Mix well by pipetting and incubate at room temperature for 2 hrs or overnight on an orbital shaker. Each sample including unknown and standard should be assayed in duplicate.
3. Wash microwell strips 3 times with 250 μL 1X Wash Buffer per well with thorough aspiration between each wash. After the last wash, empty wells and tap microwell strips on absorbent pad or paper towel to remove excess 1X Wash Buffer.
4. Add 100 μL of diluted Streptavidin-Enzyme Conjugate to each well and incubate at 37 °C for 1 hr.
5. Wash microwell strips 3 times with 250 μL 1X Wash Buffer per well with thorough aspiration between each wash. After the last wash, empty wells and tap microwell strips on absorbent pad or paper towel to remove excess 1X Wash Buffer.
6. Warm Substrate Solution to room temperature. Add 100 μL of Substrate Solution to each well, including the blank wells. Incubate at room temperature for 5 to 20 minutes on an orbital shaker.
7. Stop the enzymatic reaction by adding 100 μL of Stop Solution into each well, including the blank wells. Results should be read immediately (color will fade over time).
8. Read absorbance of each microwell on a spectrophotometer using 450 nm as the primary wave length. 5

Restrictions: For Research Use only

Handling

Handling Advice: Avoid multiple freeze/thaw cycles.

Storage: 4 °C/-20 °C

Storage Comment: Upon receipt, aliquot and store both the Reduced DNA and ARP-DNA Standards at -20°C to avoid multiple freeze/thaw cycles. Store all other components at 4°C.

References

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Mohammad, Mohamed, Zakaria, Abdul Razak, Saad: "Watermelon (Citrullus lanatus (Thunb.) Matsum. and Nakai) juice modulates oxidative damage induced by low dose X-ray in mice." in: BioMed research international, Vol. 2014, pp. 512834, (2014) (PubMed).

Zafiropoulos, Tsarouhas, Tsitsimpikou, Fragkiadaki, Germanakis, Tsardi, Maravgakis, Goutzourelas, Vasilaki, Kouretas, Hayes, Tsatsakis: "Cardiotoxicity in rabbits after a low-level exposure to diazinon, propoxur, and chlorpyrifos." in: Human & experimental toxicology, Vol. 33, Issue 12, pp. 1241-52, (2014) (PubMed).

Messaoudi, Gautier, Kthiri, Lelandais, Mihoub, Joseleau-Petit, Caldas, Bohn, Tolosa, Rao, Tao, Landoulsi, Bouloc, Richarme: "Global stress response in a prokaryotic model of DJ-1-associated Parkinsonism." in: Journal of bacteriology, Vol. 195, Issue 6, pp. 1167-78, (2013) (PubMed).

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Target Details

Background: Free radicals and other reactive species are constantly generated in vivo and cause oxidative damage to biomolecules, a process held in check only by the existence of multiple antioxidant and repair systems as well as the replacement of damaged lipids and proteins. DNA is probably the most biologically significant target of oxidative attack, and it is widely thought that continuous oxidative damage to DNA is a significant contributor to the age-related development of the major cancers, such as those of the colon, breast, rectum, and prostate. Among numerous types of oxidative DNA damage, apurinic/apyrimidinic (AP or abasic) site is one of the prevalent lesions of oxidative DNA damage. Abasic sites arise in DNA at a significant rate by spontaneous base loss as in depurination, by DNA oxidation, or by the action of DNA glycosylases. Estimates of the number of abasic sites generated per mammalian cell run as high as 50,000 to 200,000 per day. Unrepaired abasic sites inhibit topoisomerases, replication, and transcription and can be mutagenic because of bypass synthesis on nontemplated DNA.