Determination of NM Concentration

Place SNpc tissue in a plastic tube and carefully ground it. Weigh 10 mg of tissue for each sample, and place itin a 5 mL glass tube.

In each tube, add 1.5 mL of pH 7.4 phosphate buffer (50mM), shake, and centrifuge at 9000 x g for 30 mins. Discard the supernatant.

Wash with phosphate buffer and repeat once more.

Add 1.5ml of Tris buffer (50 mM, pH 7.4) solution, containing sodium dodecyl sulfate (5 mg/ml) and 0.2 mg/ml proteinase K to the pellet of each sample. Incubate the pellet by shaking in this solution for 2 hours at 37ºC.

Centrifuge the suspension of pigment at 9000 x g for 30 minutes.

Wash the pellet with 1.5 ml of NaCl solution (9 mg/ml) and 1.5 ml of water. Centrifuge at 9000 x g for 30 minutes.

Dissolve the NM residue in 1 ml of 1M NaOH at 80ºC for 1 hour.

Centrifuge this solution and transfer the supernatant into a quartz cuvette, measure the absorbance at 350 nm.

To run calibration curves dissolve known amounts of NM (ranging from 1 – 30 µg) in 1 ml of 1 M NaOH at 80ºC for 1 hour.

NM value was the average from 2-3 replicates. The final values of NM concentrations are expressed as µg/mg dry tissue.