Fluorescence detection of proteins in sodium dodecyl sulfate-polyacrylamide gels using environmentally benign, nonfixative, saline solution

Steinberg, Thomas H.; Lauber, Wendy M.; Berggren, Kiera N.; Kemper, Courtenay; Yue, Stephen; Patton, Wayne F.

doi:10.1002/(sici)1522-2683(20000201)21:3<497::aid-elps497>3.0.co;2-i

Cited by 66 publications

(12 citation statements)

References 39 publications

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“…Protein Quantitation-The protein content of exosome preparations was estimated by 1D-SDS-PAGE/SYPRO ® Ruby protein staining densitometry. This method is reproducible, has a linear quantitation range over three orders of magnitude (42), and is compatible with GeLC-MS/MS (43). Briefly, 5 l sample aliquots were solubilized in SDS sample buffer (2% (w/v) sodium dodecyl sulfate, 125 mM TrisHCl, pH 6.8, 12.5% (v/v) glycerol, 0.02% (w/v) bromphenol blue) and loaded into 1 mm, 10-well NuPAGE™ 4 -12% (w/v) Bis-Tris Precast gels (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

Oncogenic H-Ras Reprograms Madin-Darby Canine Kidney (MDCK) Cell-derived Exosomal Proteins Following Epithelial-Mesenchymal Transition

Tauro

Mathias

Greening

et al. 2013

Molecular & Cellular Proteomics

169

171

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Epithelial-mesenchymal transition (EMT) is a highly con- Epithelial-mesenchymal transition (EMT)1 is a cellular process whereby otherwise sessile epithelial cells undergo a shift in plasticity and acquire the ability to disseminate (1-6). Hallmarks of EMT include diminished expression of cell-cell contact and adhesion components (e.g. E-cadherin), diminished expression of cell-matrix components, decreased expression of components involved in cell polarity, elevated expression of proteins involved in cytoskeleton remodelling (e.g. vimentin), and increased expression of various matrix metalloproteinFrom the ‡Department of Biochemistry,

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Section: Methodsmentioning

confidence: 99%

Oncogenic H-Ras Reprograms Madin-Darby Canine Kidney (MDCK) Cell-derived Exosomal Proteins Following Epithelial-Mesenchymal Transition

Tauro

Mathias

Greening

et al. 2013

Molecular & Cellular Proteomics

169

171

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“…Protein Quantitation-The protein content of the sMVs, A33-Exos, and EpCAM-Exos was estimated via one-dimensional SDS-PAGE/ SYPRO ® Ruby protein staining densitometry (26). Briefly, 5 l sample aliquots were solubilized in SDS sample buffer (2% (w/v) SDS, 125 mM Tris-HCl, pH 6.8, 12.5% (v/v) glycerol, 0.02% (w/v) bromphenol blue) and loaded into 1-mm 10-well NuPAGE™ 4 -12% (w/v) Bis-Tris Precast gels (Invitrogen).…”

Section: Cell Culture and Preparation Of Concentrated Culture Medium-mentioning

confidence: 99%

Two Distinct Populations of Exosomes Are Released from LIM1863 Colon Carcinoma Cell-derived Organoids

Tauro

Greening

Mathias

et al. 2013

Molecular & Cellular Proteomics

374

361

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Exosomes are naturally occurring biological nanomembranous vesicles (ϳ40 to 100 nm) of endocytic origin that are released from diverse cell types into the extracellular space. They have pleiotropic functions such as antigen presentation and intercellular transfer of protein cargo, mRNA, microRNA, lipids, and oncogenic potential. Here we describe the isolation, via sequential immunocapture using anti-A33-and anti-EpCAM-coupled magnetic beads, of two distinct populations of exosomes released from organoids derived from human colon carcinoma cell line LIM1863. The exosome populations (A33-Exos and EpCAM-Exos) could not be distinguished via electron microscopy and contained stereotypical exosome markers such as TSG101, Alix, and HSP70. The salient finding of this study, revealed via gel-based LC-MS/MS, was the exclusive identification in EpCAM-Exos of the classical apical trafficking molecules CD63 (LAMP3), mucin 13 and the apical intestinal enzyme sucrase isomaltase and increased expression of dipeptidyl peptidase IV and the apically restricted pentaspan membrane glycoprotein prominin 1. In contrast, the A33-Exos preparation was enriched with basolateral trafficking molecules such as early endosome antigen 1, the Golgi membrane protein ADP-ribosylation factor, and clathrin. Our observations are consistent with EpCAM-and A33-Exos being released from the apical and basolateral surfaces, respectively, and the EpCAM-Exos proteome profile with widely published stereotypical exosomes. A proteome analysis of LIM1863-derived shed microvesicles (sMVs) was also performed in order to clearly distinguish A33-and EpCAMExos from sMVs. Intriguingly, several members of the MHC class I family of antigen presentation molecules were exclusively observed in A33-Exos, whereas neither MHC class I nor MHC class II molecules were observed via MS in EpCAM-Exos. Additionally, we report for the first time in any extracellular vesicle study the colocalization of EpCAM, claudin-7, and CD44 in EpCAM-Exos. Given that The microenvironment in which a tumor originates plays a critical role in its initiation, progression, and metastasis (1-3). Recent advances have indicated that although the microenvironment provides crucial signaling to maintain tissue architecture, inhibit cell growth, and constrain the malignant phenotype, it can also promote and induce cancer (4). In addition to cancer cells, the tumor microenvironment comprises normal cells, blood cells, secreted proteins and peptides, and constituents of the extracellular matrix that actively influence cell behavior. Secreted proteins, peptides, and physiological small molecules such as soluble cytokines and chemokines are currently recognized as the main exocrine and juxtacrine factors underlying cell-to-cell communication within the tumor microenvironment and providing the metastatic niche in distant organs (5). In addition to soluble secreted proteins and peptides, most cell types also release extracellular membrane vesicles (eMVs) 1 that transfer information between cells in the microenv...

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“…The last approach uses molecules which fluorescence depends on the chemical environment in which they are. Classical examples are Nile red [10], Sypro Orange and Sypro Red [4] and Sypro Tangerine [11]. This latter approach suffers generally from a lack of sensitivity.…”

Section: Resultsmentioning

confidence: 99%

“…Ruthenium fluorescent staining was performed according to Lamanda et al [17], and Sypro tangerine staining was performed in PBS accoding to Steinberg et al [11]. The gels were imaged with a Fluorimager laser scanner, operating at 488nm excitation wavelength.…”

Section: Detection On Gelsmentioning

confidence: 97%

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Ultrafast coelectrophoretic fluorescent staining of proteins with carbocyanines

Luche

Lelong

Diemer³

et al. 2007

Proteomics

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Protein detection on SDS gels or on two dimensional gels must combine several features, such as sensitivity, homogeneity from one protein to another, speed, low cost and user-friendliness. For some applications, it is also interesting to have a non fixing stain, so that proteins can be mobilized from the gel for further use (electroelution, blotting). We show here that co-electrophoretic staining by fluorophores of the oxacarbocyanine family, and especially diheptyloxacarbocyanine, offers several positive features. The sensitivity is intermediate between the one of colloidal Coomassie blue and the one of fluroescent ruthenium complexes. Detection is achieved within one hour after the end of the electrophoretic process and does not use any fixing or toxic agent. The fluorescent SDS-carbocyanine-protein complexes can be detected either with a laser scanner with an excitation wavelength of 488nm or with a UV table operating at 302nm. Excellent sequence coverage in subsequent mass spectrometry analysis of proteolytic peptides is also achieved with this detection method.3

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Fluorescence detection of proteins in sodium dodecyl sulfate-polyacrylamide gels using environmentally benign, nonfixative, saline solution

Cited by 66 publications

References 39 publications

Oncogenic H-Ras Reprograms Madin-Darby Canine Kidney (MDCK) Cell-derived Exosomal Proteins Following Epithelial-Mesenchymal Transition

Oncogenic H-Ras Reprograms Madin-Darby Canine Kidney (MDCK) Cell-derived Exosomal Proteins Following Epithelial-Mesenchymal Transition

Two Distinct Populations of Exosomes Are Released from LIM1863 Colon Carcinoma Cell-derived Organoids

Ultrafast coelectrophoretic fluorescent staining of proteins with carbocyanines

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