8-Hydroxy-Guanosine / 8-OHG Mouse Monoclonal Antibody [Clone ID: 15A3]

CAT#: AM03160PU-N

8-Hydroxy-Guanosine / 8-OHG mouse monoclonal antibody, clone 15A3, Purified

Product Images

AM03160PU used against oxidized 8-OH-dG in ischemic Rat brain tissue (Left Panel). Center Panel: DAPI staining. Right Panel: merged. Courtesy of Dr. Yang, University of New Mexico, USA.</em>

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  DNA Damage visualized on a retinal injury model using AM03160PU. Courtesy of Dr. Rajashekhar Gangaraju, University of Indiana, Department of Opthamology, Eugene and Marilyn Glick Eye Institute.</em>

Specifications

Product Data

• Clone Name: 15A3
• Applications: ELISA, IF, IHC
• Recommended Dilution: ELISA.
  Dot Blot.
  Immunoaffinity Chromatography.
  Immunohistochemistry (1/1000).
  Immunocytochemistry.
• Reactivities: Broad
• Host: Mouse
• Isotype: IgG2b
• Clonality: Monoclonal
• Immunogen: 8-Hydroxy-Guanosine-BSA and –Casein conjugates
• Specificity: Recognizes markers of Oxidative Damage to DNA (8-Hydroxy-2'-deoxyguanosine, 8-Hydroxyguanine and 8-Hydroxyguanosine).
• Formulation: PBS containing 50% Glycerol and 0.09% Sodium Azide
  State: Purified
  State: Liquid purified Ig fraction
• Concentration: lot specific
• Purification: Affinity Chromatography on Protein G
• Conjugation: Unconjugated
• Storage: Store undiluted at 2-8°C up to one month or (in aliquots) at -20°C for longer.
  Avoid repeated freezing and thawing.
• Stability: Shelf life: one year from despatch.
• Background: DNA or RNA damage is due to environmental factors and normal metabolic processes inside the cell, that then hinder the ability of the cell to carry out its functions. There are four main types of DNA due to endogenous cellular processes and they are oxidation, alkylation, hydrolysis and mismatch of the bases. During the oxidation of bases, highly reactive chemical entities collectively known as RONS, occurs. RONS stands for reactive oxygen and nitrogen species and includes nitric oxide, superoxide, hydroxyl radical, hydrogen peroxide and peroxynitrite. Numerous studies have shown that RONS causes a variety of issues including DNA damage (1). 8-hydroxyguanine, 8-hydroxy-2’-deoxyguanonsine and 8-hydroxyguanosine are all RNA and DNA markers of oxidative damage. 8-hydroxy-2’-guanosine is produced by reactive oxygen and nitrogen species including hydroxyl radical and peroxynitrite. Specifically its high biological relevance is due to its ability to induce G to T transversions, which is one of the most frequent somatic mutations (2). 8-hydroxy-guanine has been the most frequently studied type of DNA base damage, with studies in diabetes, and cancer. Base modifications of this type arise from radical-induced hydroxylation and cleavage reactions of the purine ring (3, 4). And finally, 8-hydroxy-guanosine, like 8-hydroxy-2’-guanosine, induces a mutagenic transversion of G to T in DNA. Its role has specifically been tested in the development of diabetes, hypertension and strokes (5, 6, and 7).
• Synonyms: 8-Hydroxy-2'-deoxyguanosine, 8-Hydroxyguanine, 8-Hydroxyguanosine, 8-OFdG, 8-OHG, 8OG, 8OHG, 8OHdG
• Note: Protocol: Immunostaining with 8-OHdG Monoclonal Antibody AM03160PU (Clone 15A3)
  Tissue Preparation
  8-OHdG monoclonal antibody reacts on both 50 um frozen tissue sections and paraffin-embedded sections.
  Tissue should be dissected fresh and fixed in periodate-lysineparaformaldehyde (PLP) at 4°C overnight.
  PLP
  Heat 1 L dH₂O to 60°C.
  Add 60 g paraformaldehyde.
  Add 33 g dibasic NaPO₄.
  Cool to RT in a cold water bath.
  Add 9 g monobasic NaPO₄.
  Add 6.45 g Na-m-periodate.
  Add 41.1 g lysine (HCl salt).
  Filter and dilute to 3 L with dH₂O.
  Adjust pH to 7.6 with 1.0 N NaOH approx. (20-30 ml).
  
  Tissue prepared for frozen sectioning must be cryoprotected in a 20% glycerol-2% DMSO solution in phosphate buffer for 24-48 hours. Tissue will sink to the bottom of container when fully penetrated. This will eliminate freezing artifact from cutting.
  
  Glycerol-DMSO (for 3 L)
  2.4 L 0.1M phosphate buffer
  600 ml glycerol
  60 ml DMSO
  
  0.1 M Phosphate Buffer, pH 7.4 (for 1 L)
  1 L dH₂O
  11 g dibasic NaPO₄
  3 g monobasic NaPO₄
  After frozen sectioning, tissue should be stored in phosphate buffer with 0.08% sodium azide.
  
  Staining Sections By DAB Procedure
  Paraffin-embedded sections must be deparaffinized by sequential immersion in the following for 3 minutes each: xylene (twice), absolute ethanol (twice). Agitate gently in each solution. Proceed with the following procedure.
  1. Pretreat sections with a methanolperoxide solution to eliminate endogenous peroxidases.
  Methanol-Peroxide
  100 ml absolute methanol
  1 ml 33% H₂O₂
  Incubate sections in methanolperoxide solution for 30 minutes at RT.
  
  2. Wash sections 3 times for 10 minutes each in 0.1 M phosphate buffered saline (PBS)
  PBS, pH 7.4 (for 1 L)
  1 L dH₂O
  11 g dibasic NaPO₄
  3 g monobasic NaPO₄
  8.5 g NaCl
  
  3. Incubate sections for 1 hour in 10% normal goat serum in PBS.
  4. Incubate sections in the primary antibody for 18-24 hours at RT. Depending on the nature of the sample, a shorter incubation time may be used.
  It is recommended that a concentration range of 1-10 ug/ml be evaluated in order to determine the optimal concentration for each type of tissue sample. Dilute antibody in PBS containing 0.3% Triton X-100, 0.08% sodium azide and 2% normal goat serum.
  NOTE: A humidified chamber is necessary when staining paraffin sections. Slides should be placed flat and primary antibody applied over the section, covering it completely.
  5. Rinse sections 3 times for 10 minutes each in PBS.
  6. Incubate for 3 hours with peroxidase-conjugated goat antimouse IgG (Boehringer-Mannheim, Indianapolis, IN) diluted 1:300 in PBS with 2% normal goat serum.
  7. Rinse sections 3 times for 10 minutes each in PBS.
  8. Incubate sections for 5-10 minutes in a solution of 0.5 mg/ml 3,3’ diaminobenzidine tetrahydrochloride (DAB, Sigma, St. Louis, MO) and 0.005% hydrogen peroxide in 0.05 M tris HCl buffer, pH 7.6 plus imidazole (10 ml/110 ml Tris buffer).
  
  50 mM Tris Buffer, pH 7.6
  1 L dH₂O
  6 g Trizma base
  3 ml concentrated HCl (37%)
  
  Sodium Imidazole
  100 ml 0.1 M phosphate buffer
  0.7 g sodium imidazole
  
  9. Rinse sections 3 times for 10 minutes each in PBS.
  10. Mount free-floating sections on subbed slides and air dry.
  
  Subbing Solution
  500 ml dH₂O
  2.5 g gelatin
  0.25 g chromium potassium sulfate
  Heat to 60°C. Filter and proceed to coat slides. Once slides are air dried, sections can be mounted.
  
  11. Dehydrate mounted/paraffin sections by sequential immersion in the following for 3 minutes each: 70% ethanol, 95% ethanol, absolute ethanol, xylene. Agitate gently in each solution.
  12. Apply coverslip with Permount in a chemical fume hood.