This method can help answer key question in environmental research about Nitrapyrin and 3-nitrotyrosine.The main advantage of this technique is that it is a unique and highly sensitive method for detecting 3-nitrotyrosine on air sample filters.To collect total suspended particles or particulate matter of less than seven micron in size, first weigh a binder 3 quartz filter and secure it on a high volume air sampler.Cover the filter with a particle size selector to collect PM7 or leave the filter uncovered to collected total suspended particles.Run the air sampler at 1000 liters per minute continuously for seven days.Load the pre-filters into a size classification unit and install the size classification unit and a backup filter on the low volume air sampler.Run the air sampler at 116 litters per minute continuously for seven days to collect PM2.5 on the backup filter.When sampling finishes record the total flow volume.Weigh the filter and calculate the weight of the collected particles.Seal the filter in a plastic bag and store it at 30 degrees Celsius.Next prepare 500 microliters of 6X non-specific protease cocktail and acetate buffer and place it in a dalasis membrane with a molecular weight cutoff of 5000 daltons.Immerse the filled membrane in 1 L of the acetate buffer and stir it at 4 degrees Celsius for 24 to 36 hours, replacing the buffer every 12 hours to remove indogenous 3-Nitrotyrosine and Nitrite.Then measure the protein concentration in the dialysed protease solution with a bicinchoninic acid protein assay.After that remove a filter from the freezer and allow it to warm to room temperature while still sealed in the bag.Then punch up to five 6 millimeters wide circles from the particulate matter covered area of the filter, and place the circles in a 1.5 milliliters micro centrifuge tube.Prepare 300 microliters of acetate buffer containing 50
