Analysis of copper levels by atomic absorption

Cells were washed, collected, suspended in ultrapure water and immediately mixed with an equal volume of 2% SDS. Lysate was incubated at 90 °C for 15 min. Protein concentration was determined by bicinchoninic acid assay. Typically ∼20 μl of lysate containing 100 μg protein was combined with 20 μl of 40% nitric acid in a glass test tube and incubated at 50 °C for 20 min. The sample was combined with 360 μl of ultrapure water and debris was removed by centrifugation at 5,000 r.p.m. for 5 min. Copper concentration in the supernatant was measured using atomic absorption spectroscopy (AA-6650G, Shimadzu, Columbia, MO), and a copper/protein molar ratio was averaged for each sample in three independent experiments. For copper analysis of microsomes, cells were suspended in PBS and homogenized. Debris and nuclei were removed by centrifugation at 5,000 r.p.m. for 15 min. Microsomes were collected by centrifugation at 20,000g for 30 min. Pelleted microsomes were resuspended in 50 μl of ultrapure water and combined with an equal volume of 2% SDS, followed by the same procedure described above for cell lysates.