Table 1.

Troubleshooting table.

Problem	Possible reason	Solution
No signal	Defective secondary Ab	Follow manufacturer’s instructions to rehydrate the secondary Ab. Mix 10 µl of a 1:10,000 dilution of the secondary Ab in washing buffer and 50 µl of the developing reagent in a microplate, and read the chemiluminescence
	Not enough DNA loaded in each well	Run positive controls and load different concentrations of the purified DNA nitrone adducts
	No DNA nitrone adducts	Run a positive and a negative control in each plate. Do not incubate the nitrone adducts with Reacti-Bind DNA coating solution for >6 h
	Treatments bleach DNA nitrone adducts*	Extract the positive control under the same conditions used to extract DNA from samples
High background	Excessive amount of DNA loaded in each well	Use 25 µl of a 5–10 µg µl−1 DNA solution. Use the type of development reagent suggested in this protocol
	If purified DNA from blood or tissue, beware of hemopro- teins as contaminants	Add 10 µl of DNA extracted from cells or tissues without any treatment and 50 µl of the development reagent. If hemoproteins are present, there will be a chemiluminescent signal. Re-purify DNA sample
Poor reproducibility	Operational failures	Use multichannel micropipettes and an automatic washer. Include a positive control sample

^*The term bleaching refers here to an alteration of the epitope (the nitrone motif) by unknown chemical mechanisms with a consequent loss of Ab binding.