Box 1. Protocol for positive control sample for 2d-dige

TIMING: 2.5 h

1. Prepare cultured monolayer cells grown in a tissue culture dish with less than 80% confluency. Both normal and cancer cell lines can be used. For suspension cultured cells, please refer to our previous report⁴¹.
2. Wash out culture medium with PBS three times.
3. Pour 20 ml ice-cold 10% TCA solution onto the cells and place the dish on ice as soon as possible.
4. Incubate the cells with ice-cold 10% TCA on ice for 30 min.
5. Collect the cells into a 1.5 ml microcentrifuge tube using a cell scraper.
6. Centrifuge the tube at 694g for 5 min.
7. Completely remove and discard the solution, pour ice-cold PBS into the tube and break the pellet by gentle tapping (do not vortex the pellet). This operation should be done within 10 s. Washing with PBS may be critical because the sample pH is occasionally not recovered during later incubation with 50 mM Tris–HCl, pH 8.0, owing to the remaining TCA. The labeling efficiency of CyDye DIGE Fluor saturation dye is dramatically decreased at a pH lower than 8.
8. Centrifuge the tube at 694g for 5 min.
9. Remove and discard the solution.
10. Place the tube on ice. Add 500 l of urea lysis buffer and break the cell pellet as quickly as possible by pipetting.
11. Incubate on ice for 30 min.
12. Centrifuge the tube at 17,360g for 30 min.
13. Recover supernatant as a cellular protein fraction.
14. Measure protein concentration with a commercially available kit such as Protein Assay (BioRad, cat. no. 500-0006). Use a 5 g sample for protein labeling and 2D-PAGE. After adding 1.0 M Tris, pH 8.0, confirm that the pH is approximately 8.0 using a pH indicator.

The use of a smaller electrophoresis device such as the Ettan Dalttwelve separation unit may be helpful in finding a solution if you are not very experienced with 2D-PAGE.

Second, if you find that some problems may be due to laser microdissection and protein labeling, prepare the protein sample using a much larger area of laser microdissected tissues. Begin with 5 g of protein sample for protein labeling and 2D-PAGE. Once the 2D gel is optimized, gradually decrease the area of laser-microdissected tissues used for the protein samples. In our experience, 2D-DIGE can be run using protein samples from an area of laser microdissected tissues smaller than 1 mm². However, less protein often results in decreased reproducibility of the experiments, probably due to artificial absorption or loss of the proteins during the experiment procedures. Empirically, protein samples from a 1 mm² area of liver cancer, lung cancer, pancreatic cancer or esophageal cancer tissue led to reproducible results. However, the other tissues we did not examine may require a greater amount of tissue for 2D-DIGE. Thus, one should start performing the experiments using at least 1 mm² of tissue area first, and once the experiments are going well, consider reducing the area.