Microwave-assisted and methanol/acetic acid-free method for rapid staining of proteins in SDS-PAGE gels.

We describe a microwave-assisted, methanol and acetic acid-free, inexpensive method for rapid staining of SDS-PAGE proteins. Only citric acid, benzoic acid, and Coomassie brilliant blue G-250 (CBG) were used. Microwave irradiation reduced the detection duration, and proteins in a clear background were visualized within 30 min of destaining, after 2 min of fixing and 12 min of staining. By using this protocol, comparable band intensities were obtained to the conventional methanol/acetic acid method.

Specific glycine to alanine mutation eliminates dynamic interaction of polymeric GlyT1a N-terminus with Coomassie Brilliant Blue G-250.

We previously found that multimeric GlyT1aN16 protein exhibits increased diffusion in a polyacrylamide gel and shows an unusual time-dependent absorbance rearrangement, as revealed by the Bradford assay. Here, we find that glycine to alanine mutation eliminates the absorbance shift, but not the altered diffusion properties of GlyT1aN16, indicating that these two phenomena are not interconnected. The absorbance shift is apparent with both native and urea-denatured GlyT1aN16, suggesting that the effect is either not dependent on protein structure, or the required structure is restored very quickly following denaturant removal. In the far-UV spectra, circular dichroism (CD) curves for both wild-type and mutated GlyT1aN16 are under the zero line, indicating largely unstructured character. However, a significant shift of the mutant CD curve suggests possible microstructural heterogeneity. Deconvolution of the CD data indicates a potential 3-fold increase in isolated helical content, which would inhibit an absorbance shift, as we demonstrated previously. These results suggest that, in addition to protein quantification, Coomassie Brilliant Blue G-250 can be used to reveal certain properties of the secondary structure of proteins.

Synthesis rifaximin with copper (Rif-Cu) and copper oxide (Rif-CuO) nanoparticles Considerable dye decolorization: An application of aerobic oxidation of eco-friendly sustainable approach.

In this study, rifaximin with copper (Cu) and copper oxide (CuO) nanoparticles (NPs) were synthesised. The resultant CuO nanoparticles were used to degrade Rhodamine B (RhB) and Coomassie Brilliant Blue (G250). Rifaximin copper and copper oxide nanoparticles were characterised using Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), ultraviolet-visible spectroscopy (UV), X-ray Photoelectron Spectroscopy (XPS), Transmission Electron Microscopy (TEM), and gas chromatography-electrochemical mass spectrometry (GC-EI-MS). An FT-IR study confirmed the formation of Cu in the 562 cm-1 peak range. Rifaximin Cu and CuO Nanoparticles displayed UV absorption peaks at 253 nm and 230 nm, respectively. Coomassie Brilliant Blue G250 was completely decolourised in Cu nanoparticles at 100 %, and Rhodamine B was also decolourised in Rifaximin CuO nanoparticles at 73 %, although Coomassie Brilliant Blue G250 Rifaximin Cu nanoparticles absorbed a high percentage of dye decolorization. The aerobic oxidation of isopropanol conversion was confirmed by GC-MS analysis. Retention time of 27.35 and 30.32 was confirmed using Cu and CuO nanoparticles as the final products of 2-propanone. It is used in the textile and pharmaceutical industries for aerobic alcohol oxidation. Rifaximin CuO nanoparticles highly active in aerobic oxidation. The novelty of this study is that, for the first time, rifaximin was used for the synthesis of copper and copper oxide nanoparticles, and it successfully achieved decolorization and aerobic oxidation.

Quantitative and qualitative analysis of three DNA extraction methods from soybean, maize, and canola oils and investigation of the presence of genetically modified organisms (GMOs).

The objective of this study was to develop a DNA-based method for the identification and tracking of edible oils, which is important for health management. Three different DNA extraction methods (CTAB, MBST kit, and manual hexane-based method) were used to obtain high-purity DNA from crude and refined soybean, maize, and canola oils. PCR was then conducted using specific primers to identify the presence of genes related to each oil type and to assess transgenicity. The results showed that DNA was present in crude and refined oils, but in very low amounts. However, using method 3 for DNA extraction provided sufficient quantity and quality of DNA for successful PCR amplification. The study concluded that the main challenge in DNA extraction from oils is the presence of PCR inhibitors, which can be overcome using the manual hexane-based method. Also, the examination of protein presence in the oils using SDS-PAGE did not indicate any protein bands.

A porous form Coomassie brilliant blue G250-isorhamnetin fluorescent composite coated with acrylic resin for tumor cell imaging.

Four distinct fluorescence complexes, the fluorescent complex-1 (FC-1), fluorescent complex-2 (FC-2), fluorescent complex third (FC-3) and fluorescent complex fourth (FC-4), were created using isorhamnetin and Coomassie brilliant blue G250 as raw materials. The issue of isorhamnetin's low solubility has been resolved, and isorhamnetin-coomassie brilliant blue G250 now has better biocompatibility. Four different forms of fluorescence compounds' ultraviolet absorption spectra were identified. It was discovered that FC-2, FC-3, and FC-4, respectively, had double peaks at 483-620 nm. FC-4 had the highest ultraviolet absorption intensity, whereas FC-1 exhibited the most consistent and longest wavelength of ultraviolet absorption. Transmission electron microscopy revealed that the acrylic resin evenly disseminated the Coomassie brilliant blue G250-isorhamnetin complex in an amorphous flocculent form. Human prostate cancer cells (PC3) and human cervical cancer cells (HeLa) were investigated in the (Cell Counting Kit-8) CCK8 experiment under 10 different concentration circumstances, and the proliferation impact was 64.30% and 68.06%, respectively. Shown the complex's strong anti-tumor properties and minimal cytotoxicity. Through in vitro imaging of tumor cells, it was found that FC-1's fluorescent complex has high selectivity and can accurately infiltrate tumor cells, proving that it is biocompatible. The design not only addresses the issue of isorhamnein-Coomassie Bright Blue G250's bioavailability, but it also has an effective visual fluorescence targeting effect.

Analysis of the enzymatic degradation of lysozyme fibrils using a combination method of non-denaturing gel electrophoresis and double staining with Coomassie Brilliant Blue G-250 and R-250 dyes.

The Amyloid fibrils of proteins are involved in various diseases, such as Alzheimer's disease. To suppress such amyloid fibrils, it is essential to develop methods to elucidate their enzymatic degradation process. Lysozyme in egg white has been well studied as a model protein of amyloid fibrils. Here, we establish a method for separating and evaluating both lysozyme fibrils and their enzymatic degradation products by combining non-denaturing gel electrophoresis and anionic dye staining with Congo red and two Coomassie brilliant blue (CBB) dyes. By combining non-denaturing gel electrophoresis and amyloid-specific Congo red staining, the separation site of lysozyme fibril was stained explicitly by Congo red and identified on the gel, and the amount of lysozyme fibrils decreased following the enzymatic degradation of lysozyme fibrils. Both lysozyme fibrils and their enzymatic degradation products were separated and examined by combining non-denaturing gel electrophoresis and double staining with CBB G-250 and R-250 dyes. Protein stained with negatively charged colloidal CBB G-250 could migrate to the anode side of electrophoresis. Following gel electrophoresis, noncolloidal CBB R-250 was used to detect lysozyme fibrils and the enzymatic degradation products. This method can be applied to investigate the enzymatic degradation process of amyloid fibrils.

Effects of cold atmospheric plasma pre-treatment on maintaining the quality of ready-to-eat drunken red shrimp (Solenocera crassicornis) stored at chilled conditions.

This present study investigated the effect of cold atmospheric plasma (CAP) pre-treatment on the quality of ready-to-eat drunken red shrimp (Solenocera crassicornis) during chilled storage. The shrimp were pre-treated with the CAP at 40 kV and 36 kH for 100 s in a plasma generating equipment before the drunken treatment and compared with an untreated control sample. The results showed that the CAP pre-treatment significantly inhibited the total viable count (TVC) values, total volatile basic nitrogen (TVB-N) content, and polyphenol oxidase (PPO) activity of the drunken shrimp compared to the control treatment. Furthermore, the CAP pre-treatment also significantly maintained the myofibrillar protein (MP) content, texture properties, and a more stable histological structure of muscle fibers compared to the control. High-throughput sequencing results confirmed that the CAP pre-treatment significantly reduced the diversity and abundance of several bacteria in the shrimp. Gas chromatography-ion mobility spectrometry (GC-IMS) analysis detected that the CAP pre-treatment effectively maintained the stability of volatile organic compounds (VOCs). These findings provide valuable theoretical support for the processing and storage of drunken shrimp.

Coomassie brilliant blue G-250 dye attenuates bleomycin-induced lung fibrosis by regulating the NF-κB and NLRP3 crosstalk: A novel approach for filling an unmet medical need.

Pulmonary fibrosis (PF) is a life-threatening disorder with a very poor prognosis. Because of the complexity of PF pathological mechanisms, filling such an unmet medical need is challenging. A number of pulmonary diseases have been linked to the activation of NF-κB and the NLRP3 inflammasome. Coomassie brilliant blue G-250 (CBBG) is proved to be a safe highly selective P2×7R antagonist with promising consequent inactivation of NLRP3 inflammasome. This is the first report to investigate the effect of CBBG on the bleomycin-induced lung fibrosis in rats. Our findings revealed that CBBG resulted in a significant improvement in histological features and oxidative status biomarkers of bleomycin-exposed lung tissue. Additionally, CBBG repressed collagen deposition as indicated after the analysis of hydroxyproline, TGF-ß, PDGF-BB, TIMP-1, MMP-9, Col1a1, SMA and ICAM-1. It also exhibited anti-inflammatory potential as revealed by the determination of TNF-α, IL-1ß, IL-18, MCP-1 in the lung tissue. In the bronchoalveolar lavage, the total protein and the LDH activity were substantially reduced. The lung protective effects of CBBG might be attributed on the one hand to the inhibition of NLRP3 inflammasome and on the other hand to the inactivation of NF-κB. Decreased levels of phospho-p65 and its DNA-binding activity as well as the analysis of TLR4 confirmed NF-κB inactivation. Caspase-1 activity is suppressed as a consequence of inhibiting NLRP3 inflammasome assembly. To conclude, CBBG may act as a primary or adjuvant therapy for the management of PF and therefore it may pose an opportunity for a novel approach to an unmet medical need.

Two-Dimensional Blue Native/SDS Polyacrylamide Gel Electrophoresis for Analysis of Brazilian Bothrops Snake Venoms.

Viperidae snakes are the most important agents of snakebites in Brazil. The protein composition of snake venoms has been frequently analyzed by means of electrophoretic techniques, but the interaction of proteins in venoms has barely been addressed. An electrophoretic technique that has gained prominence to study this type of interaction is blue native polyacrylamide gel electrophoresis (BN-PAGE), which allows for the high-resolution separation of proteins in their native form. These protein complexes can be further discriminated by a second-dimension gel electrophoresis (SDS-PAGE) from lanes cut from BN-PAGE. Once there is no study on the use of bidimensional BN/SDS-PAGE with snake venoms, this study initially standardized the BN/SDS-PAGE technique in order to evaluate protein interactions in Bothrops atrox, Bothrops erythromelas, and Bothrops jararaca snake venoms. Results of BN/SDS-PAGE showed that native protein complexes were present, and that snake venom metalloproteinases and venom serine proteinases maintained their enzymatic activity after BN/SDS-PAGE. C-type lectin-like proteins were identified by Western blotting. Therefore, bidimensional BN/SDS-PAGE proved to be an easy, practical, and efficient method for separating functional venom proteins according to their assemblage in complexes, as well as to analyze their biological activities in further details.

Effect of ripening and variety on the physiochemical quality and flavor of fermented Chinese chili pepper (Paojiao).

This work monitored the effect of ripening and variety on the physiochemical quality and flavor of fermented Chinese chili pepper (Paojiao). Three commercial varieties of chili pepper (Capsicum frutescens Linn.) at three ripening stages were selected. Physiochemical quality (color, texture, and vitamin C) and flavor properties [capsaicinoids, free amino acid (FAA), and aroma] were determined and compared by multivariate data analysis. The hardness and chewiness decreased, while the contents of vitamin C, capsaicin, and taste-active FAAs increased in Paojiao with ripening. More volatiles were found in green peppers. Fingerprinting and multivariate data analysis revealed that ester, aldehydes, and terpenes were discriminant volatiles that significantly changed in Paojiao during ripening. In general, ripening and variety greatly affect the physiochemical and flavor quality of peppers and their effects intensify after fermentation.

Evaluation of the physiochemical and aromatic qualities of pickled Chinese pepper (Paojiao) and their influence on consumer acceptability by using targeted and untargeted multivariate approaches.

Paojiao, which is traditionally pickled Chinese chili peppers (Capsicum frutescens Linn.), possesses unique flavors. This study was conducted to investigate the flavor, mouthfeel, and consumer acceptability of Paojiao. The quality of six homemade and two industrial Paojiao samples were evaluated by integrating untargeted (to fingerprint volatile fraction) and targeted (to analyze free amino acids or FAAs, capsaicinoids, and texture) approaches. Furthermore, multivariate data analysis (MVDA) was applied to identify the quality characteristics that drive the consumer acceptability for Paojiao. As for the targeted quality, hardness (1.30-10.52 N) and capsaicin (2.22-3.84 mg/g) varied with different samples. The main taste-active FAAs in Paojiao were detected. A total of 127 volatiles were observed, and esters were the major components. Based on sensory analysis, the homemade samples received higher acceptability than the industrial samples. MVDA demonstrated that some key volatiles, taste-active FAAs, and chewiness were the discriminant quality attributes affecting consumer acceptability.

Characterization and antibacterial mechanism of poly(aminoethyl) modified chitin synthesized via a facile one-step pathway.

This work aims to synthesize poly(aminoethyl) modified chitin (PAEMC) and ascertain its antibacterial activity and mechanism. FTIR and 1H NMR results proved aminoethyl moieties were grafted to C6OH and C3OH on chitin backbone in the form of polymerization. XRD and TG/DTG analyses manifested its well-defined crystallinity and thermostability. PAEMC, with average molecular weight (MW) of 851.0 kDa, degree of deacetylation (DD) of 27.95%, and degree of substitution (DS) of 1.77, had good solubility in aqueous solutions over the pH range of 3-12, and also possessed high antimicrobial activity against Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Bacillus proteus, and Klebsiella pneumoniae, commonly causing chronic wound infections. Nucleic acid release, protein leakage, increased inner membrane permeability, and decreased cell surface hydrophobicity implied bacterial cytomembranes were substantially compromised in the presence of PAEMC. Microscopically, PAEMC visually perturbed bacteria, illustrating deformed and even collapsed morphologies. Overall, PAEMC possessed good solubility, effectively destroyed bacteria via a membrane damage mechanism, and might serve as an antibacterial agent for treatments of chronic wound infections.

Physiological and proteomic analyses of coix seed aging during storage.

Although a series of physio-biochemical changes of coix seed occur during the storage, the underlying mechanism remains unknown. The present study aimed to investigate the aging mechanism of coix seed during storage. Proteome patterns of coix seed stored for 1-month, 5-month and 10-month at room temperature were compared using 2-dimensional gel electrophoresis (2-DE) and mass spectra. Thirty-one differentially expressed proteins (DEPs) were detected, which involved seven pathways including starch and sucrose metabolism, carbon metabolism, RNA transport, proteasome, protein processing in endoplasmic reticulum, ribosome, and RNA degradation. Sucrose synthase 1 was associated with sucrose metabolism and affected the sucrose content during the storage. Increased ambient temperature enhanced respiration of coix after 5-month storage, and overexpression of aconitate hydratase 2 promoted the generation of energy. In addition, the proteins involved in the antioxidant system and resistance stimulus of coix seed were mostly up-regulated during storage.

Two-dimensional blue native/SDS polyacrylamide gel electrophoresis for analysis of Brazilian Bothrops snake venoms

Viperidae snakes are the most important agents of snakebites in Brazil. The protein composition of snake venoms has been frequently analyzed by means of electrophoretic techniques, but the interaction of proteins in venoms has barely been addressed. An electrophoretic technique that has gained prominence to study this type of interaction is blue native polyacrylamide gel electrophoresis (BN-PAGE), which allows for the high-resolution separation of proteins in their native form. These protein complexes can be further discriminated by a second-dimension gel electrophoresis (SDS-PAGE) from lanes cut from BN-PAGE. Once there is no study on the use of bidimensional BN/SDS-PAGE with snake venoms, this study initially standardized the BN/SDS-PAGE technique in order to evaluate protein interactions in Bothrops atrox, Bothrops erythromelas, and Bothrops jararaca snake venoms. Results of BN/SDS-PAGE showed that native protein complexes were present, and that snake venom metalloproteinases and venom serine proteinases maintained their enzymatic activity after BN/SDS-PAGE. C-type lectin-like proteins were identified by Western blotting. Therefore, bidimensional BN/SDS-PAGE proved to be an easy, practical, and efficient method for separating functional venom proteins according to their assemblage in complexes, as well as to analyze their biological activities in further details.

The analysis of the causes of protein precipitate formation in the blanched soymilk.

This paper explored the causes of protein precipitate formation in blanched soymilk prepared by blanching soybeans through studying the changes in composition and amount of protein particles during its thermal processing. Compared with the traditional method of preparing soymilk, blanching changed the thermal aggregation behavior of protein particles. Results showed that when blanching was applied to soybeans, ß-conglycinin (7S) was denatured in the blanched soybeans, which resulted in the fixation and aggregation of 7S prior to the grinding processing. Therefore, 7S lost its inhibitory ability on the growth of other protein aggregation, explaining the increased insoluble precipitates in the blanched soymilk.

An advance for removing antinutritional protease inhibitors: Soybean whey purification of Bowman-Birk chymotrypsin inhibitor by combination of two oppositely charged polysaccharides.

Two successive and selective coacervations induced by chitosan (Ch) and carrageenan (CG) were applied to remove antinutritional protease inhibitors and purify Bowman-Birk protease inhibitor (BBI) from soybean whey. At the first coacervation induced by Ch (66.7, 200, and 510kDa), only Kunitz trypsin inhibitor (KTI) and BBI complexed with Ch were extracted, while ß-amylase and soybean agglutinin remained in supernatant. The binding constants for the interaction increased on the order Ch-66.7

A Dynamic Interaction of Coomassie Dye with the Glycine Transporters N-termini.

Coomassie Brilliant Blue interacts with proteins and even though the interactions exhibit variation due to the amino acid content, reported dye interactions with individual proteins appear to be relatively stable. Here we report an atypical dynamic interaction of glycine transporters 1 and 2 N-termini with Coomassie dye, resulting in intramolecular interference with their Bradford assay. These proteins exhibit classic protein-Coomassie G-250 complex with absorption maximum at 595 nm, which within minutes starts to decrease and parallel increase of absorbance shoulders above 300 and 700 nm is observed. Interestingly, these effects are eliminated upon fusion of glycine transporters N-termini with glutathione S-transferase protein or by the presence of glutathione S-transferase or bovine serum albumin in the same solution. Circular dichroism data revealed largely unstructured character of glycine transporters N-termini, which suggests that dynamic properties of these protein- Coomassie complexes might be a signature of high flexibility and protein disorder. The assay might potentially reveal similar domains in other proteins and help to associate them with particular functions.

Yield and textural properties of tofu as affected by soymilk coagulation prepared by a high-temperature pressure cooking process.

The cooking of raw soymilk is a necessary procedure prior to the production of tofu. The effects of the high-temperature pressure cooking (HTPC) and traditional cooking methods on the yield and textural properties of tofu products were investigated. Results showed that when HTPC was applied, the content of protein particles increased, thereby contributing to the formation of a dense network of tofu gel. Thus, significant improvement of textural properties, including hardness, chewiness and springiness, was observed. Moreover, HTPC contributes to the change in the composition of the particulate protein, whereas the proportion of ß-conglycinin in the non-particulate protein increased. The start and end points of the protein coagulation induced by Ca(2+) moved backward, and slowed the coagulation process, which was conducive to the incorporation of water or dry matter into the gel.

Synthesis of a novel chitosan-based Ce(IV) complex with proteolytic activity in vitro toward edible biological proteins.

The occurrence of enzymatic activities is attributed to proper spatial organization of functional groups from first principles. A novel chitosan-based Ce(IV) complex (CC[Ce(IV)]), an artificial metalloproteinase, was synthesized by attaching cyclen, Ce(IV), and chlorophyll-Cu(II) to a chitosan-based matrix. The enzymatic hydrolytic efficiency (HE) and the procedure of catalyzing myoglobin (Mb) by CC[Ce(IV)] in vitro were investigated using spectrophotometry, electrophoresis, and liquid chromatography. The results showed that the HE of Mb was up to 60% at 60°C within 24h, displaying a catalytic proficiency. The pseudo-first-order kinetic constant (kobs) for CC[Ce(IV)] treatment within 24h was 3.85×10(-2)h(-1), higher than that for α-chymotrypsin treatment, which was 2.63×10(-2)h(-1). Moreover, the peptide bond derived from Asp-Phe/Phe-Asp in Mb could be specifically cleaved by CC[Ce(IV)], which could simulate the functionality of α-chymotrypsin. This work provides an experimental basis for potential utilization of the chitosan-based Ce(IV) complexes in the food industry.

Effects of high-temperature pressure cooking and traditional cooking on soymilk: Protein particles formation and sensory quality.

This study focused on the effect of high-temperature pressure cooking on the sensory quality of soymilk. Soymilk was prepared by high-temperature pressure cooking (105-125°C and 0.12-0.235MPa) and traditional cooking (97°C and 0.1MPa). The size distribution and composition of protein particles and the rheological properties of soymilk were compared. Results showed that the content of protein particles and the average size of soymilk particles were higher in high-temperature pressure cooking than in traditional cooking (p<0.05). High-temperature pressure cooking affected soymilk protein denaturation and favored protein aggregation. Similar to traditional soymilk, soymilk cooked at 115°C was categorized as a Newtonian fluid but was found with increased viscosity in the rheological test. Soymilk cooked at 115°C for 10min exhibited a homogeneous, smooth, and creamy texture with a high acceptability in the sensory test.