Chemidoc mp fluorescent imager

Organs (lungs, kidney, heart, liver, and brain) from CSE^–/– and CSE^+/+ mice were isolated on ice, cleaned from connective tissue, and washed with ice-cold PBS. Immediately after isolation, the tissue was minced and homogenized on ice in HEN buffer (100 mM HEPES, 1 mM EDTA, 0.1 mM neocuproine, 1% SDS, pH7.4) supplemented with 20 mM MSBTA, 1% NP40 and 1% protease inhibitor cocktail, using Potter homogenizer. Homogenates were centrifuged at 10 000 × g for 20 min at 4 °C and clear supernatant was further incubated on ice for 1 h. 200 μL of supernatant samples were precipitated with water/methanol/chloroform mixture (v/v/v: 4/4/1) and centrifuged at 14 000 × g for 20 min at 4 °C. Obtained protein precipitates were dried under vacuum at 4 °C and stored at –80 °C. For improved tag-switch assay equal amounts of sample proteins (50 μg) were incubated with 100 μM Cy3-CN in 50 mM HEPES (pH 7.4) supplemented with 1.5% SDS, in dark, at 37 °C for 2 h, mixed with 4× non-reducing sample buffer (Bio-Rad, USA) and incubated for 30 min at 50 °C in dark. Samples were resolved on 10% SDS polyacrylamide gels in dark and immediately after short fixation and washing gels were scanned using ChemiDoc MP fluorescent imager (Bio-Rad, USA). Obtained images were semi-quantified using ImageJ software (NIH, USA).