cyanine-dye-3 and cyanine-dye-2

Free flow electrophoresis (FFE) has been applied in numerous studies as a protein separation technique due to its multiple advantages such as fast and efficient sample recovery, high resolving power, high reproducibility, and wide applicability to protein classes. As a stand-alone platform however, its utility in comparative proteomic analysis is limited as protein samples must be run sequentially rather than simultaneously which introduces inherent variability when attempting to perform quantitative analysis. Here we describe an approach combining fluorescent CyDye technology (DIGE) with FFE to simultaneously separate and identify differentially expressed proteins in a model cell system.

Topics: Buffers; Carbocyanines; Cell Extracts; Denaturing Gradient Gel Electrophoresis; Densitometry; Fluorescent Dyes; HT29 Cells; Humans; Hydrogen-Ion Concentration; Image Processing, Computer-Assisted; Isoelectric Point; Proteins; Staining and Labeling

Differential in-gel electrophoresis (DIGE) experiments allow three protein samples to be run per gel. The three samples are labeled with the spectrally resolvable fluorescent dyes, Cy2, Cy3, and Cy5, respectively. Here, we show that protein-specific dye effects exist, and we present a linear mixed model for analysis of DIGE data which takes dye effects into account. A Java implementation of the model, called DIGEanalyzer, is freely available at http://bioinfo.thep.lu.se/digeanalyzer.html. Three DIGE experiments from our laboratory, with 173, 64, and 24 gels, respectively, were used to quantify and verify the dye effects. DeCyder 5.0 and 6.5 were used for spot detection and matching. The fractions of proteins with a statistically significant (0.001 level) dye effect were 19, 34, and 23%, respectively. The fractions of proteins with a dye effect above 1.4-fold change were 1, 4, and 6%, respectively. The median magnitude of the dye effect was 1.07-fold change for Cy5 versus Cy3 and 1.16-fold change for Cy3 versus Cy2. The maximal dye effect was a seven-fold change. The dye effects of spots corresponding to the same protein tend to be similar within each of the three experiments, and to a smaller degree across experiments.

Topics: Algorithms; Animals; Brain Chemistry; Breast Neoplasms; Carbocyanines; Computational Biology; Electrophoresis, Gel, Two-Dimensional; Female; Fluorescent Dyes; Humans; Image Processing, Computer-Assisted; Internet; Linear Models; Ovarian Neoplasms; Proteins; Proteomics; Rats; Software; Tandem Mass Spectrometry

Two-dimensional difference gel electrophoresis (DIGE) is a tool for measuring changes in protein expression between samples involving pre-electrophoretic labeling with cyanine dyes. Here we assess a common method to analyze DIGE data using the DeCyder software system. Experimental error was studied by a series of same sample comparisons. Aliquots of sample were labeled with N-hydroxyl succinimidyl ester-derivatives of Cy2, Cy3, and Cy5 dyes and run together on one gel. This allowed assessment of how experimental error influenced differential expression analysis. Bias in the log volume ratios was observed, which could be explained by differences in dye background. Further complications are caused by significant gel-to-gel variation in the spot volume ratio distributions. Using DeCyder alone results in an inability to define ratio thresholds for 90 or 95% confidence. An alternative normalization method was thus applied which resulted in improved data distribution and allowed greater sensitivity in analysis. When combined with a standardizing function, this allowed gel-independent thresholds for 90% confidence. The new approach, detailed here, represents a method to greatly improve the success of DIGE data analysis.

Topics: Bacterial Proteins; Bias; Carbocyanines; Confidence Intervals; Electrophoresis, Gel, Two-Dimensional; Erwinia; Fluorescent Dyes; Proteins; Reproducibility of Results; Sensitivity and Specificity; Software; Staining and Labeling

An immunohistochemical technique was developed to visualize nitric oxide synthase (NOS)-immunopositive neurons in fresh-frozen tissue sections of rat brain for laser capture microdissection (LCM) and mRNA analysis. The effect of tissue fixation and the choice of fluorophore were investigated. Here we describe a rapid immunofluorescence protocol that allows the processing of fresh-frozen tissue sections within eight minutes and subsequent mRNA extraction and real-time PCR from pools of 20 NOS-immunopositive LCM neurons. The cellular complement of a subset of ionotropic glutamate receptors, specifically N-methyl-D-aspartate receptor subunit mRNAs, was examined because these receptor complexes are thought to mediate the effects of fast and slow glutamate excitotoxicity. Real-time PCR data revealed that striatal NOS interneurons express the mRNAs encoding NR1, NR2A, NR2B, and NR2D but not NR2C. These LCM mRNA data are consistent with previous in situ hybridization studies and demonstrate the utility of rapid immuno-LCM with real-time quantitative PCR for the study of mRNA abundance in discrete populations of neurons within the mammalian brain.

Topics: Animals; Carbocyanines; Cell Separation; Computer Systems; Corpus Striatum; Fluorescent Antibody Technique, Indirect; Fluorescent Dyes; Frozen Sections; Hydrazines; Interneurons; Isoenzymes; Lasers; Nerve Tissue Proteins; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Polymerase Chain Reaction; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Rhodamines; RNA, Messenger; Specimen Handling; Time Factors