Molecular characterisation of SGCE-associated myoclonus-dystonia ...

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Molecular characterisation of SGCE-associated myoclonus-dystonia ...

Patients, material and methods 37

To study consequences of oxidative stress several fibroblast cultures of Family W were

treated with the superoxide generator paraquat (0.5mM for 48h).

2.3.6 Mitochondrial preparation

Mitochondria were isolated from fibroblasts as previously described by Almeida and Medina

(1997). Cells were removed from flasks by incubating the cells with 0.5% (wt/vol) trypsin

resuspended in versene for 5min. Trypsinisation was stopped by addition of an equal volume

of isolation medium supplemented with 10% (vol/vol) foetal bovine serum. The cells were

pelleted by centrifugation at 1000rpm for 5min. (The pellet can be frozen at -80°C at this

point.) To proceed, cells were resuspended in 1ml ice-cold homogenization buffer. Cells were

then centrifuged at 4000 x g for 5min and the supernatant was discarded. The pellet was again

resuspended in 2ml ice-cold homogenisation buffer. Cells were optimally homogenised on ice

by 20 strokes of a tight fitting glass-teflon homogeniser revolving at 1000rpm. Cell

homogenates were centrifuged at 1500 x g for 10min at 4 ºC, the supernatant placed on ice,

and the pellet resuspended in 2ml homogenisation buffer, homogenised and centrifuged as

above. The supernatants were then combined and centrifuged once more at 1500 x g (10min,

4ºC). The pellet was discarded, and the supernatant centrifuged at 11500 x g for 12min at 4ºC.

The received mitochondria pellet was resuspended in 75μl homogenization buffer, frozen in

liquid nitrogen, and stored at –80ºC until required. The activity of the mitochondrial marker

enzyme citrate synthase was enriched approximately 3-fold between the initial cell

homogenate and the final mitochondrial pellet.

2.3.7 Protein determination

Sample protein concentration was determined by use of the BCA protein assay kit according

manufacture’s protocol. Absorbance was measured on a plate reader at 562nm. Sample

protein concentration was calculated from the BCA standard calibration curve (0-1500μg/ml).

2.3.8 Mitochondrial enzyme assays

The activities of complexes I to IV, of citrate synthase and of malate dehydrogenase were

determined in mitochondrial preparations by means of a spectrophotometical approach. The

data of the complexes were expressed against citrate synthase.

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