Two-dimensional electrophoresis of proteins has preceded, and accompanied, the birth of proteomics. Although it is no longer the only experimental scheme used in modern proteomics, it still has ...distinct features and advantages. The purpose of this tutorial paper is to guide the reader through the history of the field, then through the main steps of the process, from sample preparation to in-gel detection of proteins, commenting the constraints and caveats of the technique. Then the limitations and positive features of two-dimensional electrophoresis are discussed (e.g. its unique ability to separate complete proteins and its easy interfacing with immunoblotting techniques), so that the optimal type of applications of this technique in current and future proteomics can be perceived. This is illustrated by a detailed example taken from the literature and commented in detail. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP 2).
Two-dimensional gel electrophoresis has been instrumental in the birth and developments of proteomics, although it is no longer the exclusive separation tool used in the field of proteomics. In this ...review, a historical perspective is made, starting from the days where two-dimensional gels were used and the word proteomics did not even exist. The events that have led to the birth of proteomics are also recalled, ending with a description of the now well-known limitations of two-dimensional gels in proteomics. However, the often-underestimated advantages of two-dimensional gels are also underlined, leading to a description of how and when to use two-dimensional gels for the best in a proteomics approach. Taking support of these advantages (robustness, resolution, and ability to separate entire, intact proteins), possible future applications of this technique in proteomics are also mentioned.
This review article starts with the birth of 2D electrophoresis, investigates how it has been instrumental to the birth of proteomics, and examines its position in the current proteomics landscape. Display omitted
Sulfidation is a key process for silver nanoparticles released from consumer products in the environment. This study focuses on the impact of a model soil microorganism, Bacillus subtilis, on the ...fate of pristine and already sulfidized Ag-NPs. The nanoparticles were incubated with the initial growth medium, isolated secretome, and living bacteria, and characterized for their size and morphology, agglomeration state, structure, and Ag speciation. No Ag internalization or sorption on the cell wall was detected. A partial sulfidation, leading to an Ag–Ag2S core–shell structure, was observed in the presence of the secretome, and the rate limiting step of the reaction was the oxidation of Ag0, and it was favored near the crystal dislocations. The sulfidation was complete in the presence of the living bacteria and followed an indirect pathway. Both crystalline Ag2S and amorphous Ag2S and/or Ag-thiol were identified. At the opposite, the bacteria had no impact on Ag2S. These results suggest that microorganisms participate in the sulfidation of Ag-NPs in aerobic systems such as unsaturated soils, and thus affect the bioavailability of Ag. It is important to take these transformations into account during exposure experiments, since they drastically change the exposure conditions. Finally, the secretome of B. subtilis might be used for the green synthesis of Ag–Ag2S core–shell nanoparticles.
Silver nanoparticles (Ag-NPs) adhered/inserted on textile fibers have an effective antimicrobial role. However, their release due to low adherence and their fate in the natural settings have been ...questioned in terms of toxicity level. In order to overcome this recurrent problem of adherence, the in situ formation of Ag-NPs in five textile fibers (cotton (untreated and chemically bleached), sheep’s wool, polyamide, and polyester) was assessed. Herein, the fibers were first immersed in a silver ion solution (1 g/L of AgNO3) for ion saturation at room T for 24 h followed by draining fibers and their reimmersion this time in a strong chemical reducing solution (0.25 g/L of NaBH4) at room T for 24 h. This latter step leads to the in situ formation of Ag-NPs where size (5 nm < size < 50 nm), surface covering concentration, and aggregation degree depend on the textile fiber kind as deduced from FESEM images. This simple lab chemical method allows instantaneous in situ formation of Ag-NPs onto fibers without the requirement of additional thermal treatment. Moreover, for natural fibers, the formation of Ag-NPs inside of them is also expected as confirmed from FESEM images in cotton cross sections. In complement, all textile fibers containing Ag-NPs (sheep’s wool 10 mg/g > untreated cotton 2.3 mg/g > bleached cotton 1 mg/g > polyamide 0.62 mg/g > polyester 0.28 mg/g) were submitted to interact with strong oxidants in an aqueous media (7.5% v/v of H2O2, 0.5 and 0.05 M of HNO3 and ultrapure water as the control) using flow-through reactor experiments. Here, breakthrough curves reveal that the oxidative dissolution rate (given in mol/g min) of adhered Ag-NPs (ionic release) depends strongly on fiber nature, and nature and concentration of oxidant solution. In summary, this fundamental study suggests that Ag-NPs may be successfully adhered/inserted in natural fibers (wool and cotton) in a safety-design perspective with performant biocide properties as confirmed by using Bacillus subtilis.
Due to the physicochemical properties of nanoparticles, the use of nanomaterials increases over time in industrial and medical processes. We herein report the negative impact of nanoparticles, using ...solid growth conditions mimicking a biofilm, on the ability of Bacillus subtilis to fight against a stress. Bacteria have been exposed to sublethal doses of nanoparticles corresponding to conditions that bacteria may meet in their natural biotopes, the upper layer of soil or the gut microbiome. The analysis of the proteomic data obtained by shotgun mass spectrometry have shown that several metabolic pathways are affected in response to nanoparticles, n-ZnO or n-TiO.sub.2, or zinc salt: the methyglyoxal and thiol metabolisms, the oxidative stress and the stringent responses. Nanoparticles being embedded in the agar medium, these impacts are the consequence of a physiological adaptation rather than a physical cell injury. Overall, these results show that nanoparticles, by altering bacterial physiology and especially the ability to resist to a stress, may have profound influences on a "good bacteria", Bacillus subtilis, in its natural biotope and moreover, on the global equilibrium of this biotope.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
For many years, silver nanoparticles, as with other antibacterial nanoparticles, have been extensively used in manufactured products. However, their fate in the environment is unclear and raises ...questions. We studied the fate of silver nanoparticles in the presence of bacteria under growth conditions that are similar to those found naturally in the environment (that is, bacteria in a stationary phase with low nutrient concentrations). We demonstrated that the viability and the metabolism of a gram-positive bacteria, Bacillus subtilis, exposed during the stationary phase is unaffected by 1 mg/L of silver nanoparticles. These results can be partly explained by a physical interaction of the poly-gamma-glutamate (PGA) secreted by Bacillus subtilis with the silver nanoparticles. The coating of the silver nanoparticles by the secreted PGA likely results in a loss of the bioavailability of nanoparticles and, consequently, a decrease of their biocidal effect.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Proteomics can be defined as the large-scale analysis of proteins. Due to the complexity of biological systems, it is required to concatenate various separation techniques prior to mass spectrometry. ...These techniques, dealing with proteins or peptides, can rely on chromatography or electrophoresis. In this review, the electrophoretic techniques are under scrutiny. Their principles are recalled, and their applications for peptide and protein separations are presented and critically discussed. In addition, the features that are specific to gel electrophoresis and that interplay with mass spectrometry (i.e., protein detection after electrophoresis, and the process leading from a gel piece to a solution of peptides) are also discussed.
The quality and ease of proteomics analysis depends on the performance of the analytical tools used, and thus of the performances of the protein separation tools used to deconvolute complex protein ...samples. Among protein samples, membrane proteins are one of the most difficult sample classes, because of their hydrophobicity and embedment in the lipid bilayers. This review deals with the recent progresses and advances made in the separation of membrane proteins by 2-DE separating only denatured proteins. Traditional 2-D methods, i.e., methods using IEF in the first dimension are compared to methods using only zone electrophoresis in both dimensions, i.e., electrophoresis in the presence of cationic or anionic detergents. The overall performances and fields of application of both types of method is critically examined, as are future prospects for this field
Two-dimensional gel electrophoresis was instrumental in the birth of proteomics in the late 1980s. However, it is now often considered as an outdated technique for proteomics-a thing of the past. ...Although this opinion may be true for some biological questions, e.g., when analysis depth is of critical importance, for many others, two-dimensional gel electrophoresis-based proteomics still has a lot to offer. This is because of its robustness, its ability to separate proteoforms, and its easy interface with many powerful biochemistry techniques (including western blotting). This paper reviews where and why two-dimensional gel electrophoresis-based proteomics can still be profitably used. It emerges that, rather than being a thing of the past, two-dimensional gel electrophoresis-based proteomics is still highly valuable for many studies. Thus, its use cannot be dismissed on simple fashion arguments and, as usual, in science, the tree is to be judged by the fruit.
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