Ion procedure. These proteins might stick to the membranes of the

Ion procedure. These proteins might stick to the membranes of the isolated PBMC cells and may not have been washed away sufficiently enough. In this way, their high difference in abundance between the different samples can be linked to the sample preparation procedure. For that reason, it would also be interesting to know if proteins are suffering more from the sample preparation procedure, and are thus showing a high MedChemExpress 498-02-2 technical variation. In order to determine the technical variation, we performed two tests. As we were only interested in the variation values of the subset of protein spots used in the previous variation experiment, just these raw data were extracted. In the first test, the variance during labeling and electrophoresis was established. Herefore, a PBMC fraction from one healthy volunteer was subdivided in three samples, and each of them were labeled with Cy2, Cy3 or Cy5 respectively. After analysis of CV values of the spots, it seemed that the electrophoresis CI 1011 chemical information procedure and labeling is quite consistent during the whole experiment, as 92 of the proteins did have CV values below 20 . In the past, de Roos et al. established the within and between laboratory variation in PBMCs using classical 2D gel electrophoresis. They found a technical variation (within laboratory) that ranged from 18 to 68 [14]. Through our results, we can confirm that with the use of an internal standard in the DIGE procedure leads to less technical variation than compared to classical 2D PAGE and that the data across the gels are more comparable [25]. Furthermore, by using automated spotpickingsystems, the excision of protein spots of interest is more precise than the manual cutting. The variation due to electrophoresis and labeling 15481974 of samples is thus only a very small fraction of the total variation and has reached the optimal conditions. In a second part, the technical variance linked to the isolation of the blood cells and sample preparation was established. Herefore, six distinct PBMC isolation procedures from a single blood withdrawal were performed. Three samples were labeled with Cy3, three others with Cy5, and a pool of all 6 samples was used as internal standard and labeled with Cy2. Again, the same spots as in the previous procedure were used to calculate the CV values. After analysis, it seemed that some proteins showed a high CV (up to 99 ), and thus, have a high technical variance. This indicates that sample preparation is the most crucial step in the whole procedure and has to be optimized as much as possible. When analyzing the contribution of the technical issues due to sample preparation to the total variation (figure 3D), the majority of the spots with high total variance values, also showed a substantial contribution of technical CV. For example, the outlier with an overall variation of 148 , has a technical variation (sample preparation) of 100 . The majority of the protein spots are situated above the 45u line and thus have higher total variation values than technical variation issues. Some other protein spots which are positioned close to the 45u line, do show the same variance levels when analyzing different individuals (total CV) or the same individual (technical CV). For that reason, the isolation of PBMCs should be handled with extra care. Sufficient wash steps are required to remove the plasma proteins that stick to the leukocyte cell membrane, in order to decrease the overall variance. Furthermore it is known that several sa.Ion procedure. These proteins might stick to the membranes of the isolated PBMC cells and may not have been washed away sufficiently enough. In this way, their high difference in abundance between the different samples can be linked to the sample preparation procedure. For that reason, it would also be interesting to know if proteins are suffering more from the sample preparation procedure, and are thus showing a high technical variation. In order to determine the technical variation, we performed two tests. As we were only interested in the variation values of the subset of protein spots used in the previous variation experiment, just these raw data were extracted. In the first test, the variance during labeling and electrophoresis was established. Herefore, a PBMC fraction from one healthy volunteer was subdivided in three samples, and each of them were labeled with Cy2, Cy3 or Cy5 respectively. After analysis of CV values of the spots, it seemed that the electrophoresis procedure and labeling is quite consistent during the whole experiment, as 92 of the proteins did have CV values below 20 . In the past, de Roos et al. established the within and between laboratory variation in PBMCs using classical 2D gel electrophoresis. They found a technical variation (within laboratory) that ranged from 18 to 68 [14]. Through our results, we can confirm that with the use of an internal standard in the DIGE procedure leads to less technical variation than compared to classical 2D PAGE and that the data across the gels are more comparable [25]. Furthermore, by using automated spotpickingsystems, the excision of protein spots of interest is more precise than the manual cutting. The variation due to electrophoresis and labeling 15481974 of samples is thus only a very small fraction of the total variation and has reached the optimal conditions. In a second part, the technical variance linked to the isolation of the blood cells and sample preparation was established. Herefore, six distinct PBMC isolation procedures from a single blood withdrawal were performed. Three samples were labeled with Cy3, three others with Cy5, and a pool of all 6 samples was used as internal standard and labeled with Cy2. Again, the same spots as in the previous procedure were used to calculate the CV values. After analysis, it seemed that some proteins showed a high CV (up to 99 ), and thus, have a high technical variance. This indicates that sample preparation is the most crucial step in the whole procedure and has to be optimized as much as possible. When analyzing the contribution of the technical issues due to sample preparation to the total variation (figure 3D), the majority of the spots with high total variance values, also showed a substantial contribution of technical CV. For example, the outlier with an overall variation of 148 , has a technical variation (sample preparation) of 100 . The majority of the protein spots are situated above the 45u line and thus have higher total variation values than technical variation issues. Some other protein spots which are positioned close to the 45u line, do show the same variance levels when analyzing different individuals (total CV) or the same individual (technical CV). For that reason, the isolation of PBMCs should be handled with extra care. Sufficient wash steps are required to remove the plasma proteins that stick to the leukocyte cell membrane, in order to decrease the overall variance. Furthermore it is known that several sa.