application example

10 µl of control mouse brain and a human spinal ventral horn were extracted in 25 volumes of PBS, diluted 1+1 in a sample buffer have been loaded per well and separated on 15% SDS-PAGE and blotted for 1h to PVDF. Blots were blocked immediately following transfer in 5% Dry milk for 1h at room temperature with agitation. Blots were incubated in the primary antibody at a dilution of 1: 75 000 (AS09 535) for 1h at room temperature with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed 5 times for 5 min in TBS-T at room temperature with agitation. Blots were incubated in secondary antibody (goat anti-rabbit IgG horse radish peroxidase conjugated, from DAKO PO448) diluted to 1:42 000 for 1h at room temperature with agitation. The blots were washed as above and developed for 5 min with ECL detection reagent according to the manufacturers instructions. Images of the blots were obtained using a CCD imager. Exposure time was 30-600 seconds.