Oxidative damage to DNA is a result of the interaction of DNA with reactive oxygen species (ROS), in particular, the hydroxy radical which is converted from superoxide and hydrogen peroxide by the Fenton reaction. Hydroxy radicals produce a multiplicity of modifications in DNA. Oxidative attack by hydroxy radical on the deoxyribose moiety will lead to the release of free bases from DNA, generating strand breaks with various sugar modifications and simple AP sites. In fact, they are one of the major types of damage generated by ROS. It has been estimated that endogeneous ROS can result in about 2x10^5 base lesions per cell per day.

Aldehyde Reactive Probe (ARP) reagent (N'-aminooxymethylcarbonylhydrazino-D-biotin) reacts specifically with an aldehyde group which is the open ring form of the AP sites. This reaction makes it possible to detect DNA modifications that result in the formation of an aldehyde group. After treating DNA containing AP sites with ARP reagent, AP sites are tagged with a biotin residue. By using an excess amount of ARP, all AP sites can be converted to biotin-tagged AP sites. Therefore, AP sites can be quantified using avidin-biotin assay followed by a colorimetric detection of peroxidase or alkaline phosphatase conjugated to the avidin.

DNA Damage Quantification Kit is for quantification of abasic sites in genomic DNA, which corresponds to colorimetric 96-well microplate assay. ARP standard DNA solutions in this kit are prepared by heat/acid depurination (Lindahl & Nyberg, 1972, Kubo, et al, 1992) of calf thymus DNA to control the number of abasic sites from 0 to 40 per 1x10^5 bp. These DNA solutions are treated with ARP and purified. Standard 0 APP-DNA is prepared by methoxyamine-treated calf thymus DNA without heat/acid depurination. Since this kit contains centrifugal filtration tubes for the purification of ARP-labeled sample DNA, there is no need to determine the DNA concentration after the ARP reaction.

Standard ARP DNA and purified ARP-treated sample DNA are fixed on the 96-well plate with DNA Binding Solution. The number of the abasic sites in the sample DNA can then be determined by the biotin-avidin-peroxidase assay. Since the standard ARP DNAs are double stranded, this kit is not applicable to the abasic sites detection of single stranded DNA. The O.D. value of a single stranded DNA sample is nearly twice as high as that of a double stranded DNA sample under the same assay condition. For the isolation of genomic DNA from samples, a guanidine/detergent-based DNA isolation method is recommended.

Mechanism of ARP Tagging at an Abasic Site

Typical Calibration Curve Prepared by ARP-DNA Standard Solutions

Product Code : DK02-10 ( 5 samples ) 

Storage : 0-5 ºC

Shipping Condition : Ambient Temp.

Contents of the kit
 
   ARP Solution: 100 µl x 1 vial
   ARP-DNA Solutions: 250 µl each
   DNA Binding Solution: 10 ml x 1 bottle
   Substrate Solution: 10 ml x 1 bottle
   TE Buffer: 15 ml x 1 bottle
   HRP-Streptavidin: 1 vial
   Washing Buffer (powder for 1 L): 1 packet
   Filtration Tube: 5 tubes
   96-well Microplate / U bottom: 1 plate
   Manual: 1 booklet

Product Code : DK02-12 ( 20 samples ) 

Storage : 0-5 ºC

Shipping Condition : Ambient Temp.

 Contents of the kit

   ARP Solution: 250 µl x 1 vial
   ARP-DNA Solutions: 250 µl each
   DNA Binding Solution: 10 ml x 1 bottle
   Substrate Solution: 10 ml x 1 bottle
   TE Buffer: 15 ml x 2 bottles
   HRP-Streptavidin: 1 vial
   Washing Buffer (powder for 1 L): 1 packet
   Filtration Tube: 20 tubes
   96-well Microplate / U bottom: 1 plate
   Manual: 1 booklet

DNA Damage Quantification Kit Technical Manual in Adobe Acrobat pdf format is available

Superoxide dismutase (SOD), which catalyzes the dismutation of the superoxide anion (O₂·^-) into hydrogen peroxide and molecular oxygen, is one of the most important antioxidative enzymes. In mammals, cytosolic SOD has a greenish color and consists of two subunits: One subunit contains copper and the other zinc (Cu/Zn-SOD). Mitochondrial and bacterial SOD has a reddish-purple color and contains manganese (Mn-SOD). E. coli has Mn-SOD and Fe-SOD.

Several direct and indirect methods have been developed to determine SOD activity. An indirect method using nitrotetrazolium blue is often used because of its convenience. However, there are several disadvantages to this method, such as poor water solubility of the formazan dye and its reaction with the reduced form of xanthine oxidase. Though cytochrome C is also commonly used for SOD activity detection, its reactivity with superoxide is too high to determine low levels of SOD activity.

SOD Assay Kit-WST allows very convenient and highly sensitive SOD assay by utilizing Dojindo's highly water-soluble tetrazolium salt, WST-1 (2-(4-iodophenyl)- 3-(4-nitrophenyl)- 5-(2,4-disulfo-phenyl)- 2H-tetrazolium, monosodium salt), which produces a water-soluble formazan dye upon reduction with a superoxide anion. WST-1 is 70 times less reactive with superoxide anion than cytochrome C; therefore, highly sensitive SOD detection is possible and samples can be diluted with buffer to minimize background problems. WST-1 does not react with the reduced form of xanthine oxidase; therefore, even 100% inhibition with SOD is detectable. The rate of WST-1 reduction by superoxide anion is linearly related to the xanthine oxidase activity, and is inhibited by SOD (see figure below). Therefore, the IC₅₀ (50% inhibition concentration) of SOD or SOD-like materials can be determined using colorimetric methods (patent filing).

Advantages:
- Only kit to measure 100% inhibition by SOD
- pH independent IC50 determination
- Convenient 96-well microplate colorimetric assay
- Low-background noise measurement

Product Code : S311-10

Units : 500 tests                      

Storage : 0-5 ºC

Shipping Condition : Ambient Temp.

Mechanism of SOD Assay

SOD Assay Kit-WST Technical Manual in Adobe Acrobat format available

Glutathione (GSH) is the most abundant thiol compound in animal, plant tissues, bacteria and yeast. GSH has many different roles, including protection against reactive oxygen species and the maintenance of protein thiol groups. During these processes, GSH is converted into its oxidized form, glutathione disulfide (GSSG). Since GSSG is then enzymatically reduced by glutathione reductase, GSH is the dominant form in organisms.

DTNB (5,5'-Dithiobis(2-nitrobenzoic acid)), known as Ellman's Reagent, was developed to detect thiol compounds. In 1985, Dr. M. E. Anderson suggested that the glutathione recycling system involving DTNB and glutathione reductase could be used as a highly sensitive glutathione detection method. DTNB and GSH react to generate 2-nitro-5-thiobenzoic acid and GSSG. Since 2-nitro-5-thiobenzoic acid is yellow, the GSH concentration in a sample solution can be determined by O.D. measurement at 412 nm absorbance. GSH is regenerated from GSSG by glutathione reductase, and will again react with DTNB to produce 2-nitro-5-thiobenzoic acid. This recycling reaction improves the sensitivity of total glutathione detection.

Total Glutathione Quantification Kit contains all of the necessary reagents for total glutathione measurement, except for those used in sample preparation. 5-Sulfosalicylic acid is recommended for the removal of proteins from sample solutions and for the prevention of GSH oxidation and g-glutamyl transpeptidase reactions. However, the optimum method for sample preparation differs from sample to sample, so please review the references. This kit can be used to quantify total glutathione concentrations from 1 µM to 100 µM using the standard method. For lower glutathione concentrations, such as in blood samples, longer incubation times are required.

Advantages:
- Detection limit: 1 µmol/L
- Convenient 96-well plate colorimetric assay
- DTNB-based highly sensitive recycling system
- Short detection time (30 min)

Product Code : T419-10

Units : 100 tests                      

Storage : 0-5 ºC

Shipping Condition : Ambient Tem.

Mechanism of the Total Gluathione Quantification

Total Glutathione Quantification Kit Technical Manual in Adobe Acrobat format available

8-Nitroguanosine is a nitrated base of DNA and RNA. It is formed by peroxynitrite, which is generated from nitric oxide and superoxide anion radical. It is known that a large amount of nitric oxide molecules and superoxide anion, generated by inflammation, causes nitration of guanosine. Since chemically modified nucleotides cause mutation during DNA replication, 8-nitroguanosine is thought to be one of the markers of DNA damage related to mutation and cancer. Because of its very high specificity, monoclonal antibody NO2G52 recognizes 8-nitroguanine and 8-nitroguanosine, but it does not cross-react with normal nucleotide bases, 8-hydroxyguanine, 8-hydroxydeoxy-guanosine, 3-nitrotyrosine, xanthine or 2-nitroimidazole. The specificity of NO2G52 was determined by a competitive ELISA using an 8-nitroguanosine-BSA-coated plate. As shown in the figures below, NO₂G52 has very high affinity for 8-nitroguanine and 8-nitroguanosine, and it slightly cross-reacts with 8-bromoguanosine, 8-bromoguanine and 8-chloroguanine. Anti-Nitroguanosine polyclonal antibody also recognizes 8-nitroguanine and 8-nitroguanosine, but it does not cross-react with normal guanosine, guanine, 8-hydroxyguanine or 3-nitrotyrosine. Since this antibody was prepared using rabbits, it can be used for immuno-histostaining of rodent tissues such as the mouse or rat.

Applications

- ELISA (1 ug/ml)

- Immunohistochemistry (10 µg/ml)

Immunoreactivity [IC50]

- Strongly reacts (10 umol/l)
   8-NO₂-guanosine
   8-NO₂-guanine

- Slightly cross-reacts ( >1 mmol/l)
   8-Br-guanosine
   8-Br-guanine
   8-Cl-guanine

- No cross-reaction
guanosine, guanine, 8-OH-guanine, 8-OH-deoxyguanosine, xanthine, adenine, adenosine, thymine, deoxythymidine, uracil, uridine, 3-NO₂-tyrosine, 2-NO₂-imidazole, cytosine

Host animal

- Mouse (BALB/c)

- Isotype: IgG1

 recommended secondary antibody: anti-mouse IgG (gamma), double staining grade

Appearance and storage

- Solution
  (1 mg/ml PBS solution; 0.1% ProClin as a preservative)

- Store at -20 C

8-Nitroguanosine is a nitrated base of DNA and RNA. It is formed by peroxynitrite, which is generated from nitric oxide and superoxide anion radical. It is known that a large amount of nitric oxide molecules and superoxide anion, generated by inflammation, causes nitration of guanosine. Since chemically modified nucleotides cause mutation during DNA replication, 8-nitroguanosine is thought to be one of the markers of DNA damage related to mutation and cancer. Because of its very high specificity, monoclonal antibody NO2G52 recognizes 8-nitroguanine and 8-nitroguanosine, but it does not cross-react with normal nucleotide bases, 8-hydroxyguanine, 8-hydroxydeoxy-guanosine, 3-nitrotyrosine, xanthine or 2-nitroimidazole. The specificity of NO2G52 was determined by a competitive ELISA using an 8-nitroguanosine-BSA-coated plate. As shown in the figures below, NO₂G52 has very high affinity for 8-nitroguanine and 8-nitroguanosine, and it slightly cross-reacts with 8-bromoguanosine, 8-bromoguanine and 8-chloroguanine. Anti-Nitroguanosine polyclonal antibody also recognizes 8-nitroguanine and 8-nitroguanosine, but it does not cross-react with normal guanosine, guanine, 8-hydroxyguanine or 3-nitrotyrosine. Since this antibody was prepared using rabbits, it can be used for immuno-histostaining of rodent tissues such as the mouse or rat.

Applications

- ELISA (5 ug/ml)

- Immunohistochemistry (10 ug/ml)

Immunoreactivity [IC50]

- Strongly reacts (1 umol/l)
   8-NO₂-guanosine
   8-NO₂-guanine

- No cross-reaction
   guanosine, guanine, 8-OH-guanine, 3-NO₂-tyrosine

Host animal

- Rabbit (Japanese White)

Appearance and storage

- Solution
  (200 ug/ml PBS solution; 0.1% ProClin as a preservative)

- Store at -20 C

8-Nitroguanine is a nitrated base of DNA and RNA. It is formed by peroxynitrite, which is generated from nitric oxide and superoxide anion radical. It is known that a large amount of nitric oxide molecules and superoxide anion, generated by inflammation, causes nitration of guanosine. Since chemically modified nucleotides cause mutation during DNA replication, 8-Nitroguanine is thought to a marker of DNA damage related to mutation and cancer. 8-Nitroguanine (lyophilized) is made by the lyophilization of its phosphate buffered saline solution, and is used in immunohistochemistry for absorption testing. Adding 0.4 ml of distilled water to the 8-Nitroguanine powder produces a 1.2 mmol/l of 8-Nitroguanine solution. 8-Nitroguanine/PBS solution is stable for one month at 4 ?C. If an antibody pre-treated with excessive 8-Nitroguanine shows negative staining, then the subsequent positive staining with this antibody will be specific for 8-nitroguanine or 8-nitroguanosine formed in DNA or RNA

Product Code : N455-10

Units : 100 ug                    

Storage : 0-5 ºC

Shipping Condition : Blue Ice or Ambient Temp.

3-Deoxyglucosone (3-DG) is one of the Maillard reaction products and a very important precursor to advanced glycation end products such as pyralline, pentosidine, pyropyridine and imidazolone. 3-DG is generated by the amadori rearrangement reaction and self oxidation of fluctose. It is soluble in water.

Product Code : D535-10

Units : 10mg                    

Storage : -20ºC

Shipping Condition : Blue Ice or Dry Ice