Analysis Note

ControlNutrient starved HeLa or Cos7 cells

Application

Anti-8-Oxoguanine Antibody, clone 483.15 detects level of 8-Oxoguanine & has been published & validated for use in ELISA & IC.

Immunocytochemistry:
On HeLa and Cos7 cells fixed with paraformaldehyde. 8-oxoguanine has been localized to the nucleus in nutrient-deprived cells.

ELISA:
Cell grown in slides were extracted twice for 30 sec with cold 560nM NaCl;0.1% (v/v) Triton X-100;0.02% (w/v) SDS;10mM phosphate buffer pH 7.4 and fixed with freshly prepared 4% PFA for 5-20 minutes at room temperature. {Conlon, KA et al (2000) J. Histotechnology 23(1):37-44}. Typical staining shows that nuclei from extracted cells have define periphery and areas of condensed chromatin. Clone 483.15 staining showed specific but faint nuclear fluorescence staining in cells incubated in supplemented DMEM, but in cells incubated in nutrient-free defined solt solution (NFDSS) {1.8mM calcium chloride, 110mM NaCl, 44mM sodium biocarbonate, pH 7.5} showed strong nuclear fluorescence staining that appeared punctate and gernerally distributed. Fluorescence staining disappears to background levels in cells incubated in nutrient medias even after initial NFDSS treatments {Conlon et al}.

A previous lot of this antibody was used in an ELISA assay.

Optimal working dilutions must be determined by end user.

General description

8-Oxoguanine is a mutagenic oxidative damage product of guanine. Oxidatively damaged base 8-oxoguanine is generated in the DNA of all living organisms due to the presence of reactive oxygen species in cells. DNA damage threatens the health of the genetic information stored in every DNA molecule; however enzymes exist in cells to protect against the mutagenic effect of this lesion. 8-oxoguanine is one of the most abundant and well-characterized DNA lesions generated by oxidative stress. It has been estimated that ~180 guanines are oxidized to 8-oxoG per mammalian cell per day. 8-oxoG is a miscoding lesion that can cause G:C to T:A or T:A to G:C transversion mutations. This lesion accumulates in DNA with age and it has been loosly linked to several cancers and diseases, such as Alzheimer′s and Parkinson′s.

Immunogen

8-oxoguanine adsorbed on to alumina

Legal Information

CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany

Other Notes

Concentration: Please refer to the Certificate of Analysis for the lot-specific concentration.

Physical form

Unpurified mouse monoclonal IgM liquid in buffer containing no preservative.

Quality

Routinely evaluated by immunocytochemistry on NIH/3T3 cells..

Immunocytochemistry:
Confocal fluorescent analysis of NIH/3T3 cells using anti-8-oxoguanine mouse monoclonal antibody.

Specificity

8-oxoguanine. By competitive ELISA the monoclonal showed no reactivity with dGMP, dAMP, dCMP or TMP in micromolar concentrations. Competition was only observed when the concentrations were increased to the millimolar range.

Storage and Stability

Stable for 6 months at -20°C in undiluted aliquots from date of receipt.
Handling Recommendations: Upon receipt, and prior to removing the cap, centrifuge the vial and gently mix the solution. Aliquot into microcentrifuge tubes and store at -20°C. Avoid repeated freeze/thaw cycles, which may damage IgM and affect product performance.