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DNA Damage Quantification Colorimetric Kit

based on 7 citations in multiple journalsDNA Damage Quantification Colorimetric Kit74.2 4
Detects AP sites
Catalog #: K253
$515.00

Product Details

Cat # +Size K253-25
Size 25 assays
Detection Method Absorbance (450 and 650 nm)
Species Reactivity Mammalian
Applications Kit provides all necessary reagents for convenient determination of DNA damage (abasic sites) in sample DNA
Features & Benefits • Kit provides all necessary reagents for 96-well plate format
Kit Components • ARP Solution
• TE Buffer
• Glycogen Solution
• 0 ARP-DNA Standard
• 40 ARP-DNA Standard
• DNA Binding Solution
• HRP-Streptavidin
• 10X Wash Buffer
• HRP Developer
• 96-well Microplate (8 x 12 strips)
Storage Conditions -20°C
Shipping Conditions Gel Pack
USAGE For Research Use Only! Not For Use in Humans.

Details

Apurinic/apyrimidinic (AP) sites are one of the major types of DNA lesions formed duringthe course of base excision and repair of oxidized, deaminated or alkylated bases. It hasbeen estimated that about 2x105 base lesions are generated per cell per day. The level ofAP sites in cells can be a good indicator of DNA lesion and repair against chemicaldamage and cell aging. The DNA Damage Quantification Kit utilizes the ARP (AldehydeReactive Probe) reagent that reacts specifically with an aldehyde group which is the openring form of the AP sites. After treating DNA containing AP sites with ARP reagents, APsites are tagged with biotin residues, which can be quantified using avidin-biotin assayfollowed by a colorimetric detection. The kit provides the necessary reagents forconvenient determination of abasic sites in purified DNA sample in 96-well plate format.


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How do we prepare the DNA to be used with this kit?
We have the kit - K281-50 (Genomic DNA Isolation Kit) with which DNA can be prepared from most sample types. This DNA can then be used as a starting material with kit K253-25.
I'd like to know if ARP assay could be performed on frozen tissue samples.
The Cat # K253-25 is a DNA Damage Quantification Kit which is to be used with purified DNA. You cannot use frozen tissues directly with this kit. The frozen tissue will need pre-processing to isolate the DNA. We recommend using our kit Cat # K281-50 (Genomic DNA isolation kit) for this purpose.
When setting up analyses of DNA from genomic DNA samples it states that the concentration should be 0.1 microgram per microlitre. We have some samples that are about 10 times less concentrated and wondered if it was possible to adjust any of the volumes to take this into account?
Yes, it is essential that the DNA start conc is 0.1 µg/µl. We ask you to resuspend your genomic DNA after isolation in that conc. If you have a more dilute version, I would recommend you to concentrate the DNA to the recommended conc, by doing an agarose extraction, or any such approach. Failing this, the protocol will have to be completely modified for the various reagent volumes and you might need to do some optimization steps.
Did I understood correctly, that the biotin-labeled-DNA binds to the microplate due to interactions to avidin? Can you tell me what the Binding-Solution contains. Also in the instruction is written, that for the binding to the microplate there is an incubations time over night. Is there the opportunity to shorten this incubation time?
The DNA-ARP-Biotin binds to the plate with the help of the DNA binding solution. This solution does not have any Avidin. I am sorry, but I cannot disclose the components of the DNA binding buffer due to proprietary restrictions. The binding would be most efficient when incubated O/N, I would not recommend shortening this time.
If the provided 96-microplate in the kit is a normal once and what are the standards consisting of
The standards consist of DNA which has already been labeled with ARP, at either 0 or 40 sites/10^5 bps.
We have purchased this DNA damage kit and can I get some raw data of standard curve (absorbances of different standards) . As I am planning to test it first in my samples (with conc less than 0.1 ug/ul by using more volume than recommended). I wanted to compare if my samples are falling within the range of standards.
It’s not a good idea for your to compare your sample readings with our standards. I would recommend you to make your own standards to see if your sample readings fall within its linear range.
Can this kit be used with samples like bacteria, plants, drosophila, yeast etc?
We have optimized the kit with mammalian samples. However, theoretically these kits should work with samples from multiple species/sources. Since the optimal conditions depend on the sample type, the protocol has to be be adapted to fit the samples for efficient results. Please refer to this kit's citations to see what kind of samples have been used with this kit other than mammalian samples.
Can we use frozen samples with this assay?
Fresh samples are always preferred over frozen samples. However, frozen samples can also be used, provided, they were frozen right after isolation, were not freeze thawed multiple time (for which we recommend aliquoting the samples before freezing) and have been frozen for relatively short periods.
Can we use a different wavelength than recommended for the final analysis?
It is always recommended to use the exact recommended wavelength for the most efficient results. However, most plate readers have flexibility in their band width of detection in increments of +/- 10 nm. Depending on this flexibility range, you can deviate from the recommended wavelengths within limits.
What is the exact volume of sample required for this assay?
There is no specific volume we can recommend for the amount any sample to be used since it is completely sample concentration and quality based. You have to do a pilot expt with multiple sample volumes to determine the optimal volume which gives a reading within the linear range of the standard curve. Please refer to the citations for this product to see what other clients have used with similar sample types.
Do you have trial sizes of this kit?
Unfortunately, we do not have trial sizes of this kit available. However, if you are based in the US or Canada, we will give you a 10% off list price introductory discount on its purchase price. If you are based out of this area please contact yopur regional BioVision distributor.
What is the shelf life of this kit?
This kit is good for 12 months from the date of shipment in the unopened form when stored at the appropriate temperature and appropriate conditions. After opening and reconstitution, some of the components in this kit are good for 2 months at -20°C. Please refer to the datasheet for storage information and shelf life of each of the components.
Why are my standard curve values lower than those shown on the datasheet?
There are multiple factors which influence the signals like the incubation times, room temperature, handling etc. In general, to increase the value of the standards, you can increase the incubation time. As long as the standard curve is linear, it should be fine to use, since all of your samples will also be measured under the same conditions on this curve.
How do I normalize my samples against protein concentration
You can use a protein quantitation assay on the supernatants you get from cell/tissue lysates or with any other liquid sample in the assay buffer.
Can we purchase individual components of this kit?
Yes, you can purchase any of the kit's components without the whole kit. Please refer to the component Cat #s mentioned on the datasheet for ordering.
Can we use an alternate buffer for sample preparation (cell lysis, sample dilutions etc)?
Our assay buffers are optimized for the reactions they are designed for. They not only contain some detergents for efficient lysis of your cells/tissue, but also contain some proprietary components required for the further reactions. Therefore, we highly recommend using the buffers provided in the kit for the best results.
Should I make a standard curve for every expt I do, or is one curve/kit enough?
Yes, I would strongly recommend you to do the standards every time you do the expt. There is always a chance that something was done differently that day and we do not want any conditions to differ between standards and samples.
Mishra et al., Mitochondrial Oxidative Stress-Induced Epigenetic Modif ications in Pancreatic Epithelial Cells. International Journal of Toxicology, Mar 2014; 33: 116 - 129.
Mishra et al., Mitochondrial Oxidative Stress-Induced Epigenetic Modifications in Pancreatic Epithelial Cells. International Journal of Toxicology, Feb 2014; 10.1177/1091581814524064.
Harradine KA, et. al. Functional genomics reveals diverse cellular processes that modulate tumor cell response to oxaliplatin. Mol Cancer Res. 2011 Feb;9(2):173-82. Epub 2010 Dec 17.
Peterson TJ et al (2009) Mol. Cancer Ther.; 8: 2015 - 2026.
Doria E. et al (2009) J. Exp. Bot.; 10.1093/jxb/ern345.
For more citations of this product click here