A number of chemicals, including polycyclic aromatic hydrocarbons (PAHs), have been shown to bind to DNA. This DNA damage can occur both early and late in the malignant process, thereby acting as an initiator and assisting in the progression of tumors. PAHs are released into the environment following incomplete combustion of organic materials. The most common sources of PAHs are from smoking and from consuming broiled or grilled foods. Human exposure to PAHs comes from various occupational, environmental, dietary and medicinal sources. Benzo[a]pyrene is a representative PAH. Antibodies to benzo[a]pyrenediol-epoxide modified DNA (BPDE-DNA) can be used to identify polycyclic aromatic hydrocarbon (PAH)-DNA adducts. Exposure to this group of compounds is believed to be carcinogenic. The monoclonal antibody 5D11 recognizes BPDE-I-DNA (PAH-DNA).
Flow cytometry, Immuno assays, Immuno fluorescence, Immuno precipitation, Paraffin sections
FC: Washed sperm was fixed in 2% paraformaldehyde and permeabilized with 0.2% triton x-100/0.1%
sodium citrate. Samples were treated with protK and RNase. To denature DNA samples were
incubated with 4n HCl. After blocking with 5% normal serum samples were incubated with mAb.
IA: plates were coated with 50 ng/well BPDE-DNA in 50mM Tris-buffer pH7.5 o/n at 4°C. Plates were
blocked 1% FCS. DNA samples, 4μg, were mixed with 5D11 and added to the well. Detection with
GtαMs-IgG-AP for 90’at 37°C.
P: 5 μm sections were RNase and prot-K treated. DNA was denatured with 4N HCl and neutralized
with 50mM Tris base. Section was blocked with 1.5% normal horse serum.
For immunohistochemistry dilutions to be used depend on detection system applied. It is recommended that users test the reagent and determine their own optimal dilutions. The typical starting working dilution is 1:10.