A conventional two-color dye-swap protocol was used for antibody microarray experiments [11,17,18,20,21]. microarray spots did not show significant changes when compared with those scanned immediately after standard microarray experiments. Conclusion Microarray slides can be preserved and rescanned multiple times using a confocal laser scanner over a period of days or weeks. Background Antibody microarray analyses of protein expression levels represent a new trend of cutting-edge proteomics research [1-6], and have been increasingly utilized in studies of normal and pathological conditions [7-22]. The technique has several distinct advantages. First, compared to the cDNA microarray analysis, antibody microarrays detect differential gene expression at AZD4573 the protein level. Although cDNA microarray Rabbit polyclonal to Complement C4 beta chain analyses have generated a large database concerning gene expression patterns, most of these studies have focused on gene expression at the mRNA level only, with the assumption that this relative mRNA levels represent the relative levels of proteins. Since there is often a poor correlation between mRNA levels and protein levels [23-28], this assumption may not be true for many of the genes. Second, the antibody microarray technique is usually more sensitive compared to gel electrophoreses. With microarray technology, protein levels can be detected in the low pg/ml range. This allows the measurement of the expression of both small and large molecular weight proteins simultaneously, regardless of their isoelectric points. In addition, some protein extraction buffers used for antibody microarray experiments contain non-denaturing detergents in order to keep the proteins in their native state. While gel electrophoreses usually AZD4573 individual denatured proteins for expression analyses, antibody microarrays can measure relative abundance of na?ve undenatured proteins. The vast majority of antibody microarray experiments conducted so far used the fluorescent dyes Cy3 and Cy5 for protein labelling. In general, fluorescent dyes are sensitive to light exposure as well as other environmental factors such as water, high temperature, alkali, and alcohol. Recent evidence indicates that even ozone levels in the laboratory atmosphere could affect fluorescent dyes on microarrays [29]. AZD4573 In order to avoid or to minimize the effects of these risk factors, it is appropriate to scan AZD4573 microarray slides instantly upon finishing the final wash and drying processes without unnecessary delays. However, no data are currently available concerning time-dependent changes of fluorescent signals on antibody microarray slides. Furthermore, the published research on repeated scans of antibody or protein microarrays is usually scant. Although repeated scans of cDNA microarrays have been conducted [30-35], it is not known whether amine-coupled fluorescent dyes on antibody/protein microarrays behave in the same way as those nucleotide-coupled fluorescent dyes on cDNA microarrays. In addition, previous studies conducted multiple scans of cDNA microarrays sequentially, usually within minutes. Therefore, it is also unknown whether prolonged intervals between scans in the range of days or weeks could change the signal intensity of fluorescent dyes on antibody or protein microarrays. Furthermore, previous rescan studies were conducted using different laser power levels or photomultiplier tube (PMT) gains rather than using a consistent setting of the laser power and PMT. It is expected that different settings of the laser power and PMT generated different signal intensities. Whether the same setting of the laser power and PMT can generate consistent intensity outcome over time has not been demonstrated. Using a confocal laser scanner to minimize photo-bleaching effects, we scanned microarrays slides at day 0, 10, 20 and 30, respectively. After regular antibody microarray experiments, the microarray slides were stored in a laboratory freezer at -20C. Fluorescent intensities of microarray spots at these time points were then quantified and compared. Our results indicate no significant changes in intensities of both Cy3 and Cy5 signals over the examined period. Results and discussion Clontech? antibody microarrays were used in this study, which had 1024 microarray spots on each slide. Among them, 6 spots were printed with fluorescence-labeled albumin and served as positive controls, whereas 4 spots were printed with non-labeled albumin and served as negative controls. For all those microarray slides, the unfavorable control spots had comparable intensities as those of background signals. These 10 control spots were not included in the final microarray spot analyses. The remaining 1014 spots on each microarray slide were followed up throughout the entire length of the study. In order to keep the same experimental condition, the following steps were taken: when a storage vial made up of the microarray slide was taken out of the freezer, it.