For analysis of exogenous proteins by western blot or radioactive labeling, neurons can be grown on 3.5-cm plastic dishes and transfected in principle as described above.
1. Cover bottom of dish with 1 mg/mL polylysine and leave overnight at room temperature.
2. Remove polylysine solution and wash 2 times for 1 hour in distilled water.
3. Add 1 mL medium (minimum essential medium [MEM] + 10% horse serum) and keep overnight in the incubator.
4. Replace medium with N2 supplemented with insulin (5 μg/mL).
5. Seed cells and allow to grow for desired time. For better survival neurons can be covered with a big coverslip with paraffin dots.
1. Collect the original medium of the cells and store in the incubator.
2. Perform the transfection as described above. Afterwards, add the original medium back and incubate the cells until needed.
For analysis, prepare a cell lysate (see below) and pool three dishes per lane on a sodium dodecyl sulfate (SDS) gel.
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