Large Gel Two-Dimensional Electrophoresis: Improving Recovery of Cellular Proteome

Inagaki, Naoyuki; Katsuta, Kazuhiro

doi:10.2174/1570164043488289

Cited by 20 publications

(16 citation statements)

References 37 publications

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“…For quantitative two-dimensional electrophoresis (2-DE), stage 2 and stage 3 neurons were metabolically labeled with the culture medium containing 13% of the normal levels of methionine and cysteine plus Pro-mix (25), using a composite gel series comprising a total 93 ϫ 103-cm combined gel system (26). For differential 2-DE, neurons were metabolically labeled as above, and protein spots separated by 2-DE gels were visualized by autoradiography.…”

Section: Methodsmentioning

confidence: 99%

“…Neurons-To identify proteins involved in neuronal polarity formation, we previously performed proteome analysis of cultured rat hippocampal neurons using a 93 ϫ 103-cm large gel 2-DE (26). We screened about 6,200 protein spots on 2-DE gels and detected 277 that were consistently up-regulated during the transition between stages 2 and 3 (24).…”

Section: Identification Of Singar1 By a Proteome Screening Of Stage 2mentioning

confidence: 99%

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Singar1, a Novel RUN Domain-containing Protein, Suppresses Formation of Surplus Axons for Neuronal Polarity

Mori

Wada

Suzuki

et al. 2007

Journal of Biological Chemistry

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Although neuronal functions depend on their robust polarity, the mechanisms that ensure generation and maintenance of only a single axon remain poorly understood. Using highly sensitive two-dimensional electrophoresis-based proteomics, we identified here a novel protein, single axon-related (singar)1/KIAA0871/RPIPx/RUFY3, which contains a RUN domain and is predominantly expressed in the brain. Singar1 expression became up-regulated during polarization of cultured hippocampal neurons and remained at high levels thereafter. Singar1 was diffusely localized in hippocampal neurons and moderately accumulated in growth cones of minor processes and axons. Overexpression of singar1 did not affect normal neuronal polarization but suppressed the formation of surplus axons induced by excess levels of shootin1, a recently identified protein located upstream of phosphoinositide-3-kinase and involved in neuronal polarization. Conversely, reduction of the expression of singar1 and its splicing variant singar2 by RNA interference led to an increase in the population of neurons bearing surplus axons, in a phosphoinositide-3-kinase-dependent manner. Overexpression of singar2 did not suppress the formation of surplus axons induced by shootin1. We propose that singar1 ensures the robustness of neuronal polarity by suppressing formation of surplus axons.Most neurons develop polarity by forming a single long axon and multiple short dendrites (1, 2). The ability of neurons to develop and maintain polarity is essential for their basic functions. The polarization processes have been extensively studied in cultured hippocampal neurons (1, 3). These cells first form several minor processes, any of which appears capable of becoming either an axon or a dendrite, during the first 12-24 h after plating (stage 2). One of the neurites then rapidly elongates to acquire axonal characteristics (stage 3), whereas the others later become dendrites (stage 4). Hippocampal neurons must use a robust internal mechanism that ensures polarization as they regenerate a single axon and multiple dendrites even when polarity is altered by axonal amputation (4, 5).Recent studies have begun to reveal molecules involved in neuronal polarization (6 -8). Localization, overexpression, and loss of function studies showed that spatially localized intracellular signals of a number of molecules, such as phosphoinositide-3-kinase (PI 3-kinase) 2 (9), phosphatidylinositol (3, 4, 5) triphosphate (10), Akt (11), glycogen synthase kinase-3␤ (11-13), collapsin response mediator protein-2 (CRMP-2) (12, 14), mPar3/mPar6/atypical protein kinase C complex (9, 15), Cdc42 (15, 16), Rap1B (16), STEF/Tiam1 (15, 17), Rac (15), adenomatous polyposis coli (18), JNK (19), and DOCK7 (20) are involved in axon specification for neuronal polarity formation. As downstream events, these pathways are thought to regulate cytoskeletal networks of actin filaments (21) and microtubules (20,22).In contrast to the progress in defining the mechanisms of axon specification, our knowledge of how neurons g...

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Section: Methodsmentioning

confidence: 99%

Section: Identification Of Singar1 By a Proteome Screening Of Stage 2mentioning

confidence: 99%

Singar1, a Novel RUN Domain-containing Protein, Suppresses Formation of Surplus Axons for Neuronal Polarity

Mori

Wada

Suzuki

et al. 2007

Journal of Biological Chemistry

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“…Multiple and long SDS-PAGE gels of different polyacrylamide concentrations can help to increases the resolution in the second dimension of 2DE (Oguri et al, 2002). For example, Inagaki N et al has used six different 24 cm narrow pH range IPG strips, including pH range of 3.5-4.5, 4.0-5.0, 4.5-5.5, 5.0-6.0, 5.5-6.7 and 6-9, for the first dimensional IEF separation and two large format (24 cm 70 cm) polyacrylamide gels (7.5% and 13.5%) for second dimensional SDS-PAGE separation (Inagaki and Katsuta, 2004). In total 12 gels, they have detected about 11,000 spots from rat neuron samples.…”

Section: Gel-based Approachesmentioning

confidence: 99%

Biomarker Discovery in Clinical Proteomics: Strategies for Exposing Low Abundant Proteins

Wang¹,

Seneviratne

2008

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Disease specific proteins are highly valuable as clinical biomarkers, which can be used in early diagnosis, monitoring disease progressions and evaluating therapies. The identification and characterization of protein biomarkers in different physiological and pathological sources of interest under diverse milieu represent a key area of clinical proteomics. However, owing to the inherent complexities and the large dynamic ranges of proteins, there are many practical issues and challenges in discovering the low-abundance, disease-specific biomarkers from heterogeneous tissues and biofluids. Thus, an integrated approach for selective protein pre-fractionation, purification and separation, is necessary to detect low-abundant biomarkers in proteomics research. This review will summarize recent advancements in clinical sample preparation for removing high abundant proteins, as well as the improved two-dimensional gel electrophoresis-and mass spectrometry-based technologies for resolving low-abundant proteins. We will also elaborate the proteomic strategies for targeting protein sub-populations with special interests, such as high molecular weight protein complexes, membrane or its associated proteins, and organelle specific proteins etc. We will emphasize the use of these strategies to interrogate the current proteomic researches in clinical biomarker discovery.

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“…To further extend the effective length of IEF strips, the use of several, overlapping, narrow range pH gradient strips can be employed (Görg et al, 2004). Recently, a large scale gel system utilizing multiple pH range IEF strips and multiple SDS/PAGE gels was proposed to resolve up to 11,000 proteins with a 1 to 10 5 dynamic range in protein concentration (Inagaki and Katsuta, 2004). New apparatuses allow the processing of 24 gels at one time, in parallel, to minimize variations between gels in quantitative proteomic analysis (Eravci et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

Application of Proteomic Techniques to Fruits and Vegetables

Song¹,

Braun

2008

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Proteomics and the technology supporting this science are proving to be invaluable in elucidating the tremendous complexity of biological processes. Among proteomic techniques, two-dimensional electrophoresis (2-DE) has been applied to resolve thousands of proteins and 2-DE has been especially useful for comparative studies between paired samples or populations. Improved protein extraction and purification protocols for recalcitrant fruits and vegetables have resulted in 2-DE techniques appearing in almost all fruit and vegetable proteomic studies currently being published. Numerous proteins involving various metabolic pathways have already been reported for tomato, pepper, strawberry, grape, banana, apple and pear. Significant improvements have been made to both gel and non-gel based proteomic research platforms. Better quantitative analyses and higher throughput proteomic technologies will further improve fruit and vegetable proteomic research and will have a significant impact on future breeding and post-harvest handling technologies to improve nutritional and eating quality.

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Large Gel Two-Dimensional Electrophoresis: Improving Recovery of Cellular Proteome

Cited by 20 publications

References 37 publications

Singar1, a Novel RUN Domain-containing Protein, Suppresses Formation of Surplus Axons for Neuronal Polarity

Singar1, a Novel RUN Domain-containing Protein, Suppresses Formation of Surplus Axons for Neuronal Polarity

Biomarker Discovery in Clinical Proteomics: Strategies for Exposing Low Abundant Proteins

Application of Proteomic Techniques to Fruits and Vegetables

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