Characterization of proteins involved in intracellular pathways distinctive for colon cancer stem cells

The pH of protein samples was adjusted to 8.5 to optimize fluorescent tagging. We have used the minimal labelling. The dyes, stored at –20 ºC, were spinned briefly to ensure that the powder is at the bottom of the tube, and leaved to warm for 5 min at room temperature without opening. This will prevent exposure of the dye to condensation which may cause hydrolysis.
Then, 25 nmol of each dye (1mM) are reconstituted in 25 μl of dimethylformamide (DMF) giving a concentration of 1 nmol/μl (stock solution). After reconstitution in DMF, the dyes will give a deep color: Cy2- yellow, Cy3-red, and Cy5-blue. The test tubes are mixed vigorously for 30 seconds to dissolve the dye and centrifuged for 30 seconds at 12000 x g. Prior to labelling, the dye stock solution is diluted with DMF to a working dye solution. A concentration of 400 pmol/μl is recommended. So the dye stock solution was spinned down and 2 μl was added to 3 μl DMF.
Protein extracts (50 μg) from four different biological replicates of CD133+ and CD133- cells obtained from four different sorting were labelled separately with 400 pmol of Cy3 and Cy5 and spinned briefly to collect the solution at the bottom of the tube. A pool composed of equal amount of all samples in analysis was prepared and labelled with Cy2 to be used as internal standard for quantitative comparisons. Labelling reactions were performed on ice in the dark for 30 min and stopped with 1 mM free Lysine (final concentration).