Effect of CyDye minimum labeling in differential gel electrophoresis on the reliability of protein identification

Hrebicek, Thomas; Dürrschmid, Karin; Auer, Norbert; Bayer, Karl; Rizzi, Andreas

doi:10.1002/elps.200600639

Cited by 21 publications

(15 citation statements)

References 25 publications

(19 reference statements)

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“…Because only one gel is used in DIGE, and the same proteins from two different protein samples co-migrate as single spots, there is no need for many replicates, making spot comparison and protein quantitation much more convenient and reliable. This makes DIGE potentially amendable for high-throughput proteomics applications [22]. …”

Section: Pre-electrophoretic Protein Stains Using Differential Gelmentioning

confidence: 99%

Standard Dyes for Total Protein Staining in Gel-Based Proteomic Analysis

Chevalier

2010

Materials

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Staining of two-dimensional gels is a primary concern in proteomic studies using two-dimensional gel electrophoresis with respect to the number of proteins analyzed, the accuracy of spot quantification and reproducibility. In this review article, the efficiency of the most widely used dyes was investigated. Visible dyes (Coomassie blue and silver nitrate), fluorescent dyes (Sypro Ruby, Deep Purple) and cyanine labeled methods were compared.

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Section: Pre-electrophoretic Protein Stains Using Differential Gelmentioning

confidence: 99%

Standard Dyes for Total Protein Staining in Gel-Based Proteomic Analysis

Chevalier

2010

Materials

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“…The level of significance among the replicates was compiled by student’s t-test set to 0.05. Since we and others observed molecular weight shifts resulting by minimal labeling [40], gels were post-stained with Flamingo Pink (Bio Rad) according to the manufactures instructions. The post-stained gels were analyzed using Image Master 6.0 (GE Healthcare).…”

Section: Methodsmentioning

confidence: 99%

Growth of Murine Splenic Tissue Is Suppressed by Lymphotoxin β-Receptor Signaling (LTβR) Originating from Splenic and Non-Splenic Tissues

Milićević¹,

Nohroudi

Friederike

et al. 2016

PLoS ONE

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Development and maintenance of secondary lymphoid organs such as lymph nodes and spleen essentially depend on lymphotoxin β-receptor (LTβR) signaling. It is unclear, however, by which molecular mechanism their size is limited. Here, we investigate whether the LTβR pathway is also growth suppressing. By using splenic tissue transplantation it is possible to analyze a potential contribution of LTβR signaling inside and outside of the implanted tissue. We show that LTβR signaling within the endogenous spleen and within non-splenic tissues both significantly suppressed the regeneration of implanted splenic tissue. The suppressive activity positively correlated with the total number of LTβR expressing cells in the animal (regenerate weights of 115 ± 8 mg in LTβR deficient recipients and of 12 ± 9 mg in wild-type recipients), affected also developed splenic tissue, and was induced but not executed via LTβR signaling. Two-dimensional differential gel electrophoresis and subsequent mass spectrometry of stromal splenic tissue was applied to screen for potential factors mediating the LTβR dependent suppressive activity. Thus, LTβR dependent growth suppression is involved in regulating the size of secondary lymphoid organs, and might be therapeutically used to eradicate tertiary lymphoid tissues during autoimmune diseases.

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“…5 664 nm) allows relative quantification of all proteins detected in the three samples. However, this labeling technique is less sensitive than silver staining [8], and the Cy2-, Cy3-, and Cy5-labels affect the protein separation in both IEF and SDS-PAGE [9], especially for small proteins. Whereas these differences can be corrected by the imaging software, the high content of unlabeled proteins separated from the labeled, and thus detected species, is a more serious problem.…”

Section: Introductionmentioning

confidence: 98%

Quantitative proteomics by fluorescent labeling of cysteine residues using a set of two cyanine‐based or three rhodamine‐based dyes

Volke

Hoffmann

2008

Electrophoresis

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Despite all remarkable progress in gel-based proteomics in recent years, there is still need to further improve quantification by decreasing the detection limits and increasing the dynamic range. These criteria are achieved best by fluorescent dyes that specifically stain the proteins either by adsorption after gel electrophoresis (in-gel staining) or covalent coupling prior to gel electrophoresis (in-solution staining). Here we report a multiplex analysis of protein samples using maleimide-activated cyanine-based (Cy3 and Cy5) and rhodamine-based dyes (Dy505, Dy535, and Dy635) to permanently label all thiol-groups of cysteine-containing proteins. The detection limits in SDS-PAGE were about 10 ng per band and even 2 ng for BSA due to its high content of cysteine residues. Thus only 5 microg protein of a mouse brain homogenate were analyzed by 2-DE. Both cyanine- and rhodamine-based dyes also stained proteins that did not contain cysteines, probably by reaction with amino groups. This side reactivity did not limit the method and might even extend its general use to proteins missing cysteine residues, but at a lower sensitivity. The dynamic range was more than two orders of magnitude in SDS-PAGE and the Dy-fluorophores did not alter the mobility of the tested proteins. Thus, a mixture of Dy505-, Dy555-, and Dy635-labeled Escherichia coli lysates were separated by 2-DE in a single gel and the three spot patterns relatively quantified.

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Effect of CyDye minimum labeling in differential gel electrophoresis on the reliability of protein identification

Cited by 21 publications

References 25 publications

Standard Dyes for Total Protein Staining in Gel-Based Proteomic Analysis

Standard Dyes for Total Protein Staining in Gel-Based Proteomic Analysis

Growth of Murine Splenic Tissue Is Suppressed by Lymphotoxin β-Receptor Signaling (LTβR) Originating from Splenic and Non-Splenic Tissues

Quantitative proteomics by fluorescent labeling of cysteine residues using a set of two cyanine‐based or three rhodamine‐based dyes

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