Introduction
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.
Cell Biolabs’ 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 96 assays, including standard curve and 50 tests for unknown samples.
Related Products
1. STA-303: OxiSelect™ Nitrotyrosine Immunoblot Kit
2. STA-305: OxiSelect™ Nitrotyrosine ELISA Kit
3. STA-308: OxiSelect™ Protein Carbonyl Immunoblot Kit
4. STA-310: OxiSelect™ Protein Carbonyl ELISA Kit
5. STA-315: OxiSelect™ Protein Carbonyl Spectrophotometric Assay
6. STA-331: OxiSelect™ MDA Immunoblot Kit
7. STA-332: OxiSelect™ MDA ELISA Kit
8. STA-334: OxiSelect™ HNE Adduct ELISA Kit
9. STA-320: OxiSelect™ Oxidative DNA Damage ELISA Kit (8-OHdG)
10. STA-325: OxiSelect™ Oxidative RNA Damage ELISA Kit (8-OHG)
Kit Components
1. Glycogen Solution (Part No. 232401): One 100 μL vial of 10 mg/mL glycogen.
2. Sodium Acetate Solution (Part No. 232402): One 1.0 mL vial of 3M Sodium Acetate, pH 5.5.
3. ARP Solution (Part No. 232403): One 250 μL vial of 10 mM ARP.
3
4. DNA High-Binding Plate (Part No. 232404): One 96-well strip plate.
5. DNA Binding Solution (Part No. 232405): One 6 mL bottle.
6. 10X Wash Buffer (Part No. 232406): One 30 mL bottle.
7. Streptavidin-Enzyme Conjugate (Part No. 310803): One 20 μL vial.
8. Substrate Solution (Part No. 310807): One 12 mL amber bottle.
9. Stop Solution (Part. No. 310808): One 12 mL bottle.
10. Reduced DNA Standard (Part No. 232407): One 1.0 mL vial of 6 μg/mL fully reduced in TE
Buffer (0 ARP/100,000 bp).
11. ARP-DNA Standard (Part No. 232408): One 400 μL vial of 6 μg/mL ARP-DNA in TE Buffer (40
ARP/100,000 bp).
Materials Not Supplied
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 0C Incubator
7. Multichannel micropipette reservoir
8. Microplate reader capable of reading at 450 nm (620 nm as optional reference wave length)
Storage
Upon receipt, aliquot and store both the Reduced DNA and ARP-DNA Standards at -20ºC to avoid
multiple freeze/thaw cycles. Store all other components at 4ºC until their expiration dates.
Preparation of Reagents
• 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.
Preparation of Standard Curve
Prepare a dilution series of ARP-DNA standards in the concentration range of 0 – 40 ARP/100,000 bp
according to table 1.
4
Tubes
ARP-DNA
Standard (μL)
Reduced DNA
Standard (μL)
TE
Buffer
(μL)
Total
Volume
(μL)
DNA
Concentration
(μg/mL)
AP Sites
per 100,000
bp
1 20 0 100 120 1 40
2 16 4 100 120 1 32
3 12 8 100 120 1 24
4 8 12 100 120 1 16
5 4 16 100 120 1 8
6 2 18 100 120 1 4
7 1 19 100 120 1 2
8 0 20 100 120 1 0
Table 1. Preparation of ARP-DNA Standards
Assay Protocol
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 of ARP solution in a
microcentrifuge tube and incubate 1 hr at 37 0C.
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 0C 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 0C for up to one year.