Image Analysis Workflow for 2-D Electrophoresis Gels Based on ImageJ

Natale, Massimo; Maresca, Bernardetta; Abrescia, Paolo; Bucci, Enrico

doi:10.4137/pri.s7971

Cited by 27 publications

(27 citation statements)

References 27 publications

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“…2) which are not found in control samples; protein expression levels were analyzed using an image analyzer and the regulation values are presented in Table 2 and Fig. 3 ( Natale et al 2011). The expressed proteins were analyzed by MALDI-TOF/MS and the results are represented in Fig.…”

Section: Resultsmentioning

confidence: 99%

Proteomic characterization and bio-informatic analysis of differentially expressed E. coli Nissle 1917 proteins with response to cocoti wine stress

et al. 2017

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The present study emphases the comparative proteomic analysis of Escherichia coli Nissle 1917 under cocoti palm wine stress and identified differentially expressed proteins. Protein samples were analyzed by 2-D, MALDI-TOF combined with MS access. In 2-D electrophoresis, eight differentially expressed proteins were identified: five up-regulated, two down-regulated and one newly expressed protein. Protein spots were digested with trypsin for MALDI-TOF-MS analysis; protein sequences were obtained from MASCOT search. Sequences were aligned with template using Swiss Model server. Phyre-2 was used to predict homology modeling, RasMol was used to analyze the modeling structures, PSVS server was utilized to validate the protein structure by Ramachandran's plot analysis, physical and chemical properties were analyzed using ProtParam server, Phylogenetic tree was constructed by Mega4. UniProt search helps to find protein functional information of differentially expressed proteins, involved in catalytic activities, regulation mechanisms, DNA damage stimulus, anti-termination and termination process, protein binding, electron transport mechanism, and cell signaling process functions. A detailed exploration of the proteins under cocoti palm wine stress have provided the composition, structure and functions of the expressed proteins for further investigation.

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Section: Resultsmentioning

confidence: 99%

Proteomic characterization and bio-informatic analysis of differentially expressed E. coli Nissle 1917 proteins with response to cocoti wine stress

et al. 2017

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“…Densitometric analysis of the bands was carried out with ImageJ image analysis software [26]. Copy number of the insert was determined from the image analysis results.…”

Section: Methodsmentioning

confidence: 99%

Point mutation of the xylose reductase (XR) gene reduces xylitol accumulation and increases citric acid production in Aspergillus carbonarius

Weyda

Lübeck

Ahring

et al. 2014

Journal of Industrial Microbiology and Biotechnology

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Aspergillus carbonarius accumulates xylitol when it grows on d-xylose. In fungi, d-xylose is reduced to xylitol by the NAD(P)H-dependent xylose reductase (XR). Xylitol is then further oxidized by the NAD+-dependent xylitol dehydrogenase (XDH). The cofactor impairment between the XR and XDH can lead to the accumulation of xylitol under oxygen-limiting conditions. Most of the XRs are NADPH dependent and contain a conserved Ile-Pro-Lys-Ser motif. The only known naturally occurring NADH-dependent XR (from Candida parapsilosis) carries an arginine residue instead of the lysine in this motif. In order to overcome xylitol accumulation in A. carbonarius a Lys-274 to Arg point mutation was introduced into the XR with the aim of changing the specificity toward NADH. The effect of the genetic engineering was examined in fermentation for citric acid production and xylitol accumulation by using d-xylose as the sole carbon source. Fermentation with the mutant strain showed a 2.8-fold reduction in xylitol accumulation and 4.5-fold increase in citric acid production compared to the wild-type strain. The fact that the mutant strain shows decreased xylitol levels is assumed to be associated with the capability of the mutated XR to use the NADH generated by the XDH, thus preventing the inhibition of XDH by the high levels of NADH and ensuring the flux of xylose through the pathway. This work shows that enhanced production of citric acid can be achieved using xylose as the sole carbon source by reducing accumulation of other by-products, such as xylitol.

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“…The resulting gels were analyzed immediately by Typhoon Trio Laser scanner, employing the preset DIGE scanning protocol (GE Healthcare). Spot volumes in each color channel were quantified using plugins available for ImageJ (NIH), using Watershed plugin (available as free download at http://bigwww.epfl.ch/sage/soft/watershed/index.html) as previously described [40]. …”

Section: Methodsmentioning

confidence: 99%

Interactions by 2D Gel Electrophoresis Overlap (iGEO): a novel high fidelity approach to identify constituents of protein complexes

2013

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BackgroundHere we describe a novel approach used to identify the constituents of protein complexes with high fidelity, using the integrin-associated scaffolding protein PINCH as a test case. PINCH is comprised of five LIM domains, zinc-finger protein interaction modules. In Drosophila melanogaster, PINCH has two known high-affinity binding partners—Integrin-linked kinase (ILK) that binds to LIM1 and Ras Suppressor 1 (RSU1) that binds to LIM5—but has been postulated to bind additional proteins as well.ResultsTo purify PINCH complexes, in parallel we fused different affinity tags (Protein A and Flag) to different locations within the PINCH sequence (N- and C-terminus). We expressed these tagged versions of PINCH both in cell culture (overexpressed in Drosophila S2 cell culture in the presence of endogenous PINCH) and in vivo (at native levels in Drosophila lacking endogenous PINCH). After affinity purification, we analyzed PINCH complexes by a novel 2D-gel electrophoresis analysis, iGEO (interactions by 2D Gel Electrophoresis Overlap), with mass spectrometric identification of individual spots of interest. iGEO allowed the identification of protein partners that associate with PINCH under two independent purification strategies, providing confidence in the significance of the interaction. Proteins identified by iGEO were validated against a highly inclusive list of candidate PINCH interacting proteins identified in previous analyses by MuDPIT mass spectrometry.ConclusionsThe iGEO strategy confirmed a core complex comprised of PINCH, RSU1, ILK, and ILK binding partner Parvin. Our iGEO method also identified five novel protein partners that specifically interacted with PINCH in Drosophila S2 cell culture. Because of the improved reproducibility of 2D-GE methodology and the increasing affordability of the required labeling reagents, iGEO is a method that is accessible to most moderately well-equipped biological laboratories. The biochemical co-purifications inherent in iGEO allow for rapid and unambiguous identification of the constituents of protein complexes, without the need for extensive follow-up experiments.

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Image Analysis Workflow for 2-D Electrophoresis Gels Based on ImageJ

Cited by 27 publications

References 27 publications

Proteomic characterization and bio-informatic analysis of differentially expressed E. coli Nissle 1917 proteins with response to cocoti wine stress

Proteomic characterization and bio-informatic analysis of differentially expressed E. coli Nissle 1917 proteins with response to cocoti wine stress

Point mutation of the xylose reductase (XR) gene reduces xylitol accumulation and increases citric acid production in Aspergillus carbonarius

Interactions by 2D Gel Electrophoresis Overlap (iGEO): a novel high fidelity approach to identify constituents of protein complexes

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