UV-Induced DNA Damage ELISA Kit

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Marke OxiSelect™
Proben DNA samples
Analytische Methode Quantitative
Nachweismethode Colorimetric
Produktmerkmale OxiSelect™ Oxidative UV-induced DNA Damage ELISA Kit (6-4PP Quantitation) is an enzyme immunoassay developed for rapid detection and quantitation of 6-4PP in any DNA samples. The quantity of 6-4PP in unknown sample is determined by comparing its absorbance with that of a known 6-4PP-DNA standard curve. Each kit provides sufficient reagents to perform up to 96 assays, including standard curve and unknown samples. Figure 1: Structures of DNA lesions induced by UV Light
  1. DNA High-Binding Plate : One 96-well strip plate.
  2. DNA Binding Solution : One 6 mL bottle.
  3. Anti-6-4PP Antibody : One 20 μL vial.
  4. Secondary Antibody, HRP Conjugate : One 50 μL vial.
  5. Assay Diluent : One 50 mL bottle.
  6. 10X Wash Buffer : One 100 mL bottle.
  7. Substrate Solution : One 12 mL amber bottle.
  8. Stop Solution (Part. No. 310808): One 12 mL bottle.
  9. 6-4PP-DNA Standard : One 100 μL vial of 25 μg/mL 6-4PP-DNA in 1X TE Buffer.
  10. Reduced DNA Standard : One 100 μL vial of 0.2 mg/mL reduced DNA in TE Buffer.
Benötigtes Material
  1. DNA samples such as cell or tissue genomic DNA
  2. DNA Extraction Kit
  3. Heating Block
  4. PBS
  5. 10 μL to 1000 μL adjustable single channel micropipettes with disposable tips
  6. 50 μL to 300 μL adjustable multichannel micropipette with disposable tips
  7. Multichannel micropipette reservoir
  8. Microplate reader capable of reading at 450 nm (620 nm as optional reference wave length)
Hintergrund Absorption of ultraviolet (UV) light produces two predominant types of DNA damage, cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts (6-4PP) (Figure 1). The result is a transition of C to T and CC to TT, which are the most frequent mutations of p53 in both human and mouse skin cancers. UV damaged DNA is usually repaired by nucleotide excision repair (NER) or base excision repair (BER). After UV exposure, cells activate p53 and stall the cell cycle for repair. If the damage is too severe, the cell will trigger apoptosis to get rid of DNA damaged, potentially mutant cells.
Applikationshinweise Optimal working dilution should be determined by the investigator.

  • Measure (6-4) photoproduct structures of DNA lesions induced by UV light
  • Quantify structures in isolated DNA in standard ELISA format

Plattentyp Uncoated
Protokoll 6-4PP-DNA standards or unknown DNA samples are first heat denatured before being adsorbed onto a 96-well DNA high-binding plate. The 6-4PPs present in the sample or standard are probed with an anti-6-4PP antibody, followed by an HRP conjugated secondary antibody. The 6-4PP content in an unknown sample is determined by comparing with a standard curve that is prepared from predetermined 6-4PP-DNA standards.
Aufbereitung der Reagenzien
  • 1X Wash Buffer: Dilute the 10X Wash Buffer Concentrate to 1X with deionized water. Stir to homogeneity.
  • Anti-6-4PP Antibody and Secondary Antibody: Immediately before use dilute the Anti-6-4PP Antibody 1:1000 and Secondary Antibody 1:1000 with Assay Diluent. Do not store diluted solutions.
  1. Extract DNA from cell or tissue samples using a commercial DNA Extraction kit or other desired method.
  2. Convert DNA sample to single-stranded DNA by incubating the sample at 95 °C for 10 minutes and rapidly chilling on ice for 10 minutes.
  3. Dilute DNA samples to 4 μg/mL in cold TE Buffer. Note: Samples with high concentrations of 6-4PP may be further diluted 2-4 fold in 4 μg/mL Reduced BSA. A titration may be performed to ensure the samples fall in the range of the standard curve.
  4. Add 50 μL of unknown DNA samples or 6-4PP-DNA standards to the wells of the DNA High- Binding plate.
  5. Add 50 μL of DNA Binding Solution to each well. Mix well by pipetting and incubate at room temperature overnight on an orbital shaker. Each DNA sample including unknown and standard should be assayed in duplicate.
  6. Remove the DNA solutions and wash twice with PBS. Blot plate on paper towels to remove excess fluid. Add 200 μL of Assay Diluent to each well and block for 1 hour at room temperature.
  7. Remove the Assay Diluent. Blot plate on paper towels to remove excess fluid.
  8. Add 100 μL of the diluted Anti-6-4PP Antibody to all wells and incubate for 1 hour at room temperature on an orbital shaker.
  9. Wash 5 times with 250 μL of 1X Wash Buffer 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.
  10. Add 100 μL of the diluted Secondary Antibody-HRP Conjugate to all wells and incubate for 1 hour at room temperature on an orbital shaker. Wash the strip wells 5 times according to step 9 above.
  11. Warm Substrate Solution to room temperature. Add 100 μL of Substrate Solution to each well, including the blank wells. Incubate at room temperature on an orbital shaker. Actual incubation time may vary from 2-30 minutes. Note: Watch plate carefully, if color changes rapidly, the reaction may need to be stopped sooner to prevent saturation.
  12. Stop the enzyme reaction by adding 100 μL of Stop Solution to each well. Results should be read immediately (color will fade over time).
  13. Read absorbance of each well on a microplate reader using 450 nm as the primary wave length. Use the Reduced DNA Standard as an absorbance blank. 5
Beschränkungen Nur für Forschungszwecke einsetzbar
Handhabung Avoid multiple freeze/thaw cycles.
Lagerung 4 °C/-20 °C
Informationen zur Lagerung Upon receipt, aliquot and store the Reduced DNA and 6-4PP-DNA Standards at -20°C to avoid multiple freeze/thaw cycles. Store all other components at 4°C.
Bilder des Herstellers
Cellular Assay (CA) image for UV-Induced DNA Damage ELISA Kit (ABIN2344969) DNA Damage Induced by UV Light. Calf thymus DNA at 0.2 mg/mL was exposed to UV light ...
ELISA image for UV-Induced DNA Damage ELISA Kit (ABIN2344969) 6-4PP-DNA Standard Curve
Produkt verwendet in: Akaike, Kuwano, Nishida, Kurokawa, Kajita, Kano, Masuda, Rokutan: "Homeodomain-interacting protein kinase 2 regulates DNA damage response through interacting with heterochromatin protein 1?." in: Oncogene, Vol. 34, Issue 26, pp. 3463-73, 2015 (PubMed).

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