Silver staining is used to detect proteins after electrophoretic separation on polyacrylamide gels. It combines excellent sensitivity (in the low nanogram range) with the use of very simple and cheap ...equipment and chemicals. It is compatible with downstream processing, such as mass spectrometry analysis after protein digestion. The sequential phases of silver staining are protein fixation, then sensitization, then silver impregnation and finally image development. Several variants of silver staining are described here, which can be completed in a time range from 2 h to 1 d after the end of the electrophoretic separation. Once completed, the stain is stable for several weeks.
The present work was focused on the synthesis and characterization of hydroxyapatite doped with low concentrations of zinc (Zn:HAp) (0.01 <
< 0.05). The incorporation of low concentrations of Zn
ions ...in the hydroxyapatite (HAp) structure was achieved by co-precipitation method. The physico-chemical properties of the samples were characterized by X-ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), Scanning Electron Microscopy (SEM), zeta-potential, and DLS and N₂-BET measurements. The results obtained by XRD and FTIR studies demonstrated that doping hydroxyapatite with low concentrations of zinc leads to the formation of a hexagonal structure with lattice parameters characteristic to hydroxyapatite. The XRD studies have also shown that the crystallite size and lattice parameters of the unit cell depend on the substitutions of Ca
with Zn
in the apatitic structure. Moreover, the FTIR analysis revealed that the water content increases with the increase of zinc concentration. Furthermore, the Energy Dispersive X-ray Analysis (EDAX) and XPS analyses showed that the elements Ca, P, O, and Zn were found in all the Zn:HAp samples suggesting that the synthesized materials were zinc doped hydroxyapatite, Ca
Zn
(PO₄)₆(OH), with 0.01 ≤
≤ 0.05. Antimicrobial assays on
and
bacterial strains and HepG2 cell viability assay were carried out.
Titanium dioxide and copper oxide nanoparticles are more and more widely used because of their catalytic properties, of their light absorbing properties (titanium dioxide) or of their biocidal ...properties (copper oxide), increasing the risk of adverse health effects. In this frame, the responses of mouse macrophages were studied. Both proteomic and targeted analyses were performed to investigate several parameters, such as phagocytic capacity, cytokine release, copper release, and response at sub toxic doses. Besides titanium dioxide and copper oxide nanoparticles, copper ions were used as controls. We also showed that the overall copper release in the cell does not explain per se the toxicity observed with copper oxide nanoparticles. In addition, both copper ion and copper oxide nanoparticles, but not titanium oxide, induced DNA strands breaks in macrophages. As to functional responses, the phagocytic capacity was not hampered by any of the treatments at non-toxic doses, while copper ion decreased the lipopolysaccharide-induced cytokine and nitric oxide productions. The proteomic analyses highlighted very few changes induced by titanium dioxide nanoparticles, but an induction of heme oxygenase, an increase of glutathione synthesis and a decrease of tetrahydrobiopterin in response to copper oxide nanoparticles. Subsequent targeted analyses demonstrated that the increase in glutathione biosynthesis and the induction of heme oxygenase (e.g. by lovastatin/monacolin K) are critical for macrophages to survive a copper challenge, and that the intermediates of the catecholamine pathway induce a strong cross toxicity with copper oxide nanoparticles and copper ions.
The analysis of secreted proteins represents a challenge for current proteomics techniques. Proteins are usually secreted at low concentrations in the culture media, which makes their recovery ...difficult. In addition, culture media are rich in salts and other compounds interfering with most proteomics techniques, which makes selective precipitation of proteins almost mandatory for a correct subsequent proteomics analysis. Last but not least, the non-secreted proteins liberated in the culture medium upon lysis of a few dead cells heavily contaminate the so-called secreted proteins preparations. Several techniques have been used in the past for concentration of proteins secreted in culture media. These techniques present several drawbacks, such as coprecipitation of salts or poor yields at low protein concentrations. Improved techniques based on carrier-assisted TCA precipitation are described and discussed in this report. These techniques have been used to analyze the secretome of myeloid cells (macrophages, dendritic cells) and enabled to analyze proteins secreted at concentrations close to 1 ng/mL, thereby allowing the detection of some of the cytokines (TNF, IL-12) secreted by the myeloid cells upon activation by bacterial products.
Copper is a transition metal essential for human life. Its homeostasis is regulated in the liver, which delivers copper to the whole body and excretes its excess outside the organism in the feces ...through the bile. These functions are regulated within hepatocytes, and the ATP7B copper transporter is central to making the switch between copper use and excretion. In Wilson disease, the gene coding for ATP7B is mutated, leading to copper overload, firstly, in the liver and the brain. To better understand the role of ATP7B in hepatocytes and to provide a smart tool for the development of novel therapies against Wilson disease, we used the CrispR/Cas9 tool to generate hepatocyte cell lines with the abolished expression of ATP7B. These cell lines revealed that ATP7B plays a major role at low copper concentrations starting in the micromolar range. Moreover, metal stress markers are induced at lower copper concentrations compared to parental cells, while redox stress remains not activated. As shown recently, the main drawback induced by copper exposure is protein unfolding that is drastically exacerbated in ATP7B-deficient cells. Our data enabled us to propose that the zinc finger domain of DNAJ-A1 would serve as a sensor of Cu stress. Therefore, these Wilson-like hepatocytes are of high interest to explore in more detail the role of ATP7B.
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.
Liver is pivotal in organism metabolism. This organ is receiving nutriments from the portal vein and then storing, metabolizing, distributing in the circulation or excreting excess and xenobiotics in ...bile. Liver architecture and hepatocyte polarization are crucial to achieve these functions. To study these mechanisms in details, relevant cell culture systems are required, which is not the case with standard 2D cell culture. Besides, primary hepatocytes rapidly de-differenciate making them inefficient in forming physiological system. Herein, we used an hepatoma-derived cell line to produce matrix-free hepatic spheroids and developed an integrated structural cell biology methodology by combining light sheet fluorescence microscopy and 3D electron microscopy to study their function and structure. Within these spheroids, hepatocytes polarize and organize to form bile canaliculi active for both organics and inorganics excretion. Besides, live imaging revealed the high dynamic of actin networks in basal membranes compared to their high stability in the apical pole that constitutes bile canaliculi. Finally, the first structure of active bile canaliculi was solved at nm resolution and showed the very high density of microvilli coming from all cells constituting the canaliculus. Therefore, this study is the first comprehensive and in-depth functional and structural study of bile canaliculi in a physiological-relevant context.
Titanium dioxide nanoparticles (TiO2-NPs) are increasingly used in consumer products for their particular properties. Even though TiO2 is considered chemically stable and insoluble, studying their ...behavior in biological environments is of great importance to figure their potential dissolution and transformation. The interaction between TiO2-NPs with different sizes and crystallographic forms (anatase and rutile) and the strong chelating enterobactin (ent) siderophore was investigated to look at a possible dissolution. For the first time, direct evidence of anatase TiO2-NP surface dissolution or solubilization (i.e., the removal of Ti atoms located at the surface) in a biological medium by this siderophore was shown and the progressive formation of a hexacoordinated titanium–enterobactin (Ti–ent) complex observed. This complex was characterized by UV–visible and Fourier transform infrared (FTIR) spectroscopy (both supported by Density Functional Theory calculations) as well as electrospray ionization mass spectrometry (ESI-MS) and X-ray photoelectron spectroscopy (XPS). A maximum of ca. 6.3% of Ti surface atoms were found to be solubilized after 24 h of incubation, releasing Ti–ent complexes in the micromolar range that could then be taken up by bacteria in an iron-depleted medium. From a health and environmental point of view, the effects associated to the solubilization of the E171 TiO2 food additive in the presence of enterobactin and the entrance of the Ti–enterobactin complex in bacteria were questioned.
Peroxiredoxins are often encountered as double spots when analysed by two-dimensional electrophoresis. The quantitative balance between these two spots depends on the physiological conditions, and is ...altered in favour of the acidic variant by oxidative stress for all the peroxiredoxins we could analyse. Using HeLa cells as a model system, we have further analysed the two protein isoforms represented by the two spots for each peroxiredoxin. The use of selected enzyme digestion and MS demonstrated that the acidic variant of all the peroxiredoxins analysed is irreversibly oxidized at the active-site cysteine into cysteine sulphinic or sulphonic acid. Thus, this acidic variant represents an inactivation form of the peroxiredoxins, and provides a useful marker of oxidative damage to the cells.
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
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