Multiplex Detection and Quantitation of Proteins on Western Blots Using Fluorescent Probes

Gingrich, Jeffrey C.; Davis, D. R.; Nguyen, Quan Huu

doi:10.2144/00293pf02

Cited by 73 publications

(57 citation statements)

References 9 publications

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“…S2). Third, we quantified antigen-bound primary antibody with a secondary antibody linked to an infrared fluorophore (not an enzyme), thereby making signal intensity linear within a large dynamic range (19). Fourth, we ran (on each quantification gel) a dilution series of cell extract and a dilution series of the calibration standard for the quantified protein (Fig.…”

Section: Resultsmentioning

confidence: 99%

Scaffold number in yeast signaling system sets tradeoff between system output and dynamic range

Thomson

Benjamin

Bush

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Although the proteins comprising many signaling systems are known, less is known about their numbers per cell. Existing measurements often vary by more than 10-fold. Here, we devised improved quantification methods to measure protein abundances in the Saccharomyces cerevisiae pheromone response pathway, an archetypical signaling system. These methods limited variation between independent measurements of protein abundance to a factor of two. We used these measurements together with quantitative models to identify and investigate behaviors of the pheromone response system sensitive to precise abundances. The difference between the maximum and basal signaling output (dynamic range) of the pheromone response MAPK cascade was strongly sensitive to the abundance of Ste5, the MAPK scaffold protein, and absolute system output depended on the amount of Fus3, the MAPK. Additional analysis and experiment suggest that scaffold abundance sets a tradeoff between maximum system output and system dynamic range, a prediction supported by recent experiments.quantitative immunoblotting | single-cell fluorescence quantification | genetic algorithmic model optimization T he Saccharomyces cerevisiae pheromone response system has been useful for understanding eukaryotic cell signaling (1-3). In haploid cells, the pheromone response system detects mating pheromone in the extracellular environment secreted by yeast of the opposite mating type. The system triggers a number of responses, including induction of gene expression, arrest in cell cycle progression, changes in morphology, and eventually, mating. The molecules and interactions by which the system operates are relatively well-understood (Fig. 1).Changes in the number of molecules per cell (hereafter called abundances) of signaling proteins can alter quantitative signaling behaviors. For example, in the pheromone response system, overexpression of Fus3 increases pheromone-induced sensitivity to cell cycle arrest (4), and overexpression of Gpa1 decreases pheromone-induced transcription (5). Similarly, changes in the abundances of MAPK cascade scaffold proteins (Ste5 in the yeast pheromone response system) ( Fig. 1) can alter system behavior. In models, when scaffold is limiting, increasing scaffold abundance first increases system output and then, eventually sequesters the associated protein kinases onto separate scaffolds, diminishing the number of fully assembled complexes and system output (6). This peak and decline behavior has been experimentally shown for Ste5 in this yeast system (7) and the Kinase Suppressor of Ras (KSR) scaffold protein in Ras signaling in Xenopus oocytes (8).Previous investigators reported focused and genome-wide inventories of pheromone system protein abundances (9-13). These measurements differed by up to 12-fold (Fig. 2) (14, 15). We thought some discrepancies might be because of differences and systematic biases in quantification methods (Discussion). We developed improved measurement methods and used these methods to quantify system proteins. We used...

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Section: Resultsmentioning

confidence: 99%

Scaffold number in yeast signaling system sets tradeoff between system output and dynamic range

Thomson

Benjamin

Bush

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…The Wnt/β-catenin signaling pathway plays a key role in regulating DG adult neurogenesis (39) and is involved in regulating hippocampal NPC proliferation upon lithium stimulation in vitro (38). To confirm the activation of this pathway upon lithium administration (54), we evaluated the amount of active β-catenin (i.e., dephosphorylated at Ser37 and Thr41) (55,56) in hippocampal lysates from WT and Ts65Dn mice using quantitative multiplex fluorescent Western blotting (57). Lithium treatment effectively increased the ratio of active β-catenin to total β-catenin in both WT and Ts65Dn hippocampi.…”

Section: Figurementioning

confidence: 99%

Lithium rescues synaptic plasticity and memory in Down syndrome mice

Contestabile¹,

Greco²,

Ghezzi³

et al. 2012

J. Clin. Invest.

145

151

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Down syndrome (DS) patients exhibit abnormalities of hippocampal-dependent explicit memory, a feature that is replicated in relevant mouse models of the disease. Adult hippocampal neurogenesis, which is impaired in DS and other neuropsychiatric diseases, plays a key role in hippocampal circuit plasticity and has been implicated in learning and memory. However, it remains unknown whether increasing adult neurogenesis improves hippocampal plasticity and behavioral performance in the multifactorial context of DS. We report that, in the Ts65Dn mouse model of DS, chronic administration of lithium, a clinically used mood stabilizer, promoted the proliferation of neuronal precursor cells through the pharmacological activation of the Wnt/ β-catenin pathway and restored adult neurogenesis in the hippocampal dentate gyrus (DG) to physiological levels. The restoration of adult neurogenesis completely rescued the synaptic plasticity of newborn neurons in the DG and led to the full recovery of behavioral performance in fear conditioning, object location, and novel object recognition tests. These findings indicate that reestablishing a functional population of hippocampal newborn neurons in adult DS mice rescues hippocampal plasticity and memory and implicate adult neurogenesis as a promising therapeutic target to alleviate cognitive deficits in DS patients.

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“…In addition, blots prepared from two-dimensional gels have been successfully probed with as many as nine antibodies simultaneously (45). An alternative approach that can detect different proteins simultaneously employs antibodies conjugated with different fluorophores and quantification of fluorescence by using a molecular imager (46). With this approach, several proteins can be detected even when bands overlap.…”

Section: Targeted Proteomics Approachesmentioning

confidence: 99%

Proteomics and the Kidney

Knepper

2002

Journal of the American Society of Nephrology

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Proteomics can be defined as "the systematic analysis of proteins for their identity, quantity, and function." The central concept is "multiplexing," i.e., simultaneous analysis of all proteins in a defined protein population, rather than investigation of one protein at a time, as in traditional biochemistry. Two major approaches have been described: (1) mass spectrometry-based approaches and (2) protein micro-array approaches. The purpose of this Science Watch article is to describe the fundamental features of these two approaches and to speculate on how proteomics will be useful in nephrology and nephrology research in the coming years.

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Multiplex Detection and Quantitation of Proteins on Western Blots Using Fluorescent Probes

Cited by 73 publications

References 9 publications

Scaffold number in yeast signaling system sets tradeoff between system output and dynamic range

Scaffold number in yeast signaling system sets tradeoff between system output and dynamic range

Lithium rescues synaptic plasticity and memory in Down syndrome mice

Proteomics and the Kidney

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