CBB G-250 Research Articles

Musa acuminata free tepal (MAFT) crop- mediated synthesis of copper oxide nanocatalyst (MAFT-CuO nanocatalyst): Investigation of aerobic oxidation and dye decolourization via eco-friendly approach

Crop development is used to address environmental problems in the industry. Environmental pollutants, particularly humble or toxic chemicals and dyes, are contaminants in industrial wastewater. Recently, green technology has become important for controlling the chemical risks associated with environmental issues. This study focused on valuable crop materials to form green catalysts that can be used to control chemical toxicity in industrial wastewater. In this study, Musa acuminata-free tepal (MAFT) crops and musa were used to mediate the formation of copper nanocatalyst for environmental pollution. The synthesised nanocatalyst was analysed by XPS, XRD, TEM, SEM, FT-IR, GC-EI-MS, and TGA-DSC. The production of Cu–O in the 512 cm−1 peak range was verified by FT-IR analysis. The XRD peaks associated with the cubic crystalline structures of copper (110) and (200) were used to confirm the Musa acuminata-free tepal CuO nanocatalyst. The SEM images of the Cu and CuO nanocatalyst in the Musa acuminata free tepal showed that the particles were almost spherical. The size of the synthesised Musa acuminata free tepal CuO nanocatalyst core-shell nanostructures was identified using HR-TEM. GC-MS investigations also verified the aerobic oxidation of the main alcohol to aldehyde, with a retention time of 30.34. The MAFT was 80 % of CBB G250 dye decolourised at 24 h, and the MAFT-CuO nanocatalyst was decolourised the CBB G250 dye at 80 % in 20 min and aerobic oxidation at the same time (dual application). Furthermore, our nanocatalyst could be effectively recycled without significant loss of catalytic activity, and the use of safe, environmentally benign MAFT-Cu and CuO nanocatalyst is considered a green chemistry approach that helps avoid wastewater treatment. MAFT-Cu and CuO nanocatalyst were successfully synthesised using Musa acuminata free tepal extract assisted by reflux sample preparation system instruments as a new, green, eco-friendly, and fast process for nanocatalyst synthesis. In the past, there have been no reported applications of MAFT-Cu and CuO nanocatalyst for decolourisation and aerobic oxidation. Nevertheless, we were able to accomplish these procedures with the help of CuO and MAFT-Cu nanocatalyst. Therefore, the most effective naturally occurring environmental catalysts for aerobic oxidation processes are MAFT-CuO nanocatalyst. They are non-toxic and environmentally benign, exhibit strong aerobic oxidation catalytic performance, and have long-lasting benefits for water quality. Additionally, they are more recoverable and biodegradable.

Simultaneous Regulation of Surface Properties and Microstructure of Graphene Oxide Membranes for Enhanced Nanofiltration Performance.

The surface properties and microstructure of graphene oxide (GO)-based membranes are both crucial for enhanced nanofiltration performance. Herein, a GO nanofiltration membrane is fabricated with regulatable surface properties and microstructure via a facile two-step impregnation in KOH and following HCl aqueous solutions. The type and number of oxygen-containing groups in GO membranes change with fewer C-O-C/C-OH and C═O but more COOH groups, and they are readily regulated by alkaline treatment time, which enables enhanced surface hydrophilicity and larger surface ζ potentials. Meanwhile, a few tiny defects are present in the GO sheets, which could increase the number of pores and decrease the length of water nanochannels. Such surface properties and microstructure together determine the excellent nanofiltration performance of the GO membranes with fast and selective water permeation, e.g., ∼99.5% rejection toward CBB G250 and flux of 56.9 ± 1.0 L m-2 h-1. This work provides insights into the design of high-performance two-dimensional laminar membranes.

Photocatalytic Degradation of Acidic and Basic Dye by ZnS and Tin-Doped ZnS Nanocatalysts

Zinc sulphide (ZnS) nanomaterials were hydrothermally prepared and doped with 2% Tin (Sn). UV–Visible, X-ray Diffraction (XRD), Energy-Dispersive X-ray (EDX), Scanning Electron Microscopy (SEM), Fourier transform Infrared (FTIR) spectroscopy and Thermogravimetric analysis (TGA) were used to characterize the produced NPs. With successful doping, the band gap of ZnS nanomaterials decreased from 3.50 to 3.10 eV. Cubic crystal structure of ZnS and Sn-ZnS nanomaterials was shown by XRD with the average particle size of 16.30 and 18.62 nm, respectively. ZnS nanomaterials were found to have spherical shape, and no change in shape was noticed with Sn-doping. Using TGA analysis, 22.8% and 21.5% weight loss was observed in Zinc sulphide and Tin-doped ZnS nanocatalyst, respectively, at heating range of 40–600 °C revealing their stability to temperature. The synthesized nanoparticles were also used for the photodegradation of Coomassie Brilliant Blue G-250 (CBB G-250) and Basic Blue-3 (BB-3) dyes in aqueous solution. For photodegradation reaction using bare ZnS and Sn-doped ZnS nanomaterials, activation energies of 8.6 kJ/mol and 32.1 kJ/mol were calculated for ZnS and Sn-ZnS NPs, respectively. First-order reaction kinetics was followed by the photodegradation of both the dyes using these NPs. About 84% and 93% degradation was observed for CBB G-250 dye using bare and Sn-doped ZnS NPs at 240 min time interval while using the same nanoparticles for the degradation of BB-3 dye the degradation reached 83% and 95% at 360 min time, respectively. Low concentration (10 ppm) of dyes, maximum dosage (0.03 g) of the catalysts and increasing temperature up to 70 °C favoured the photocatalytic degradation. Per cent degradation of both the dyes was found higher at low pH(3.5). Both bare Zinc sulphide and Sn-doped Zinc sulphide nanomaterials could be recycled for the given dyes detoxification as shown by the recyclability of the used catalysts, but the performance of Sn-ZnS was higher as compared to pure ZnS. Results of antibacterial studies revealed that the synthesized NPs were more active against E. coli in all concentration as compared to others.

Investigating TRSV Phytoalexin Infusions as a Novel Treatment for Alzheimer’s Disease Using ApoE4 Plasmid Transfections in Neuro2a Cell Complexes

Because the accumulation of amyloid plaques plays a central role in the pathogenesis of Alzheimer9s disease (AD) and is responsible for many of its neurodegenerative effects, this project aims to evaluate the effectiveness of TRSV phytoalexin infusions as a novel treatment for the disease by comparing three separate agents and determining its impact on the accumulation of amyloid plaques in mouse Neuro2a cells. The experimental model is as follows: Neuro2a cells were first thawed using incubation and centrifugation techniques. The subculturing protocol was then performed to maximize cellular viability using DPBS and TrypLE dissociation reagents. The cells were transfected with pCMV4-ApoE4 bacterial plasmid using Opti-MEM medium and Lipofectamine LTX while also being treated with phytoalexin agents. Bradford9s assay was performed on the samples using CBB G-250 reagent, and they were run through a spectrophotometer and a Python low-pass filter to generate readings. The experimental data showed that natural grape seed phytoalexin extract was the most effective treating agent, followed by curcumin extract and synthetic RDS phytoalexin. All treating agents lessened the accumulation of the amyloid plaques significantly, supporting the novel infusion protocol as a treatment for AD.

Two-Dimensional Polyacrylamide Gel Electrophoresis for Metalloprotein Analysis Based on Differential Chemical Structure Recognition by CBB Dye

In an effort to develop an analytical method capable of finding new metalloproteins, this is the first report of a new diagonal gel electrophoresis method to isolate and identify metalloproteins, based on the molecular recognition of holo- and apo-metalloproteins (metalbound and -free forms, respectively) by CBB G-250 dye and employing metal ion contaminant sweeping-blue native-polyacrylamide gel electrophoresis (MICS-BN-PAGE). The difference in electrophoretic mobilities between holo- and apo-forms was exaggerated as a result of interactions between the metalloproteins and the dye with no metal ion dissociation. The different binding modes of proteins with CBB G-250 dye, primarily related to hydrogen bonding, were confirmed by capillary zone electrophoresis (CZE) and molecular docking simulations. Due to in-gel holo/apo conversion between the first and second dimensions of PAGE, holo-metalloproteins in the original sample were completely isolated as spots off the diagonal line in the second dimension of PAGE. To prove the high efficiency of this method for metalloprotein analysis, we successfully identified a copper-binding protein from a total bacterial soluble extract for the first time.

Decolorization of Coomassie brilliant blue G-250 dye using snail shell powder by action of adsorption processes

The adsorption of CBB G-250 dye on snail shell powder was investigated in adsorption process. The effects of PH, contact time, temperature and adsorbent dose were evaluated. The maximum adsorption of CBBG-250 was achieved where the removal ratio was 86.3% at the optimum initial dye concentration 20.000mg/L, the absorbent material weight 0.02g and the contact time 30 min. The balance of adsorption and kinetics was found for Freundlich, Langmuir and Temkinisotherm. Thermal dynamics of the adsorption processΔH, ΔG and ΔS were found exothermal, spontaneous, and decrease in randomness at the solid/solution interface.

A simple and cost-effective solid-phase protein nano-assay using polyacrylamide-coated glass plates

A new solid-phase protein nano-assay is suggested for simple and sensitive estimation of protein content in sample buffers (a 1-μl sample is sufficient for analysis). The assay is different from conventional “on-filter” assays in that it uses inexpensive fully transparent polyacrylamide gel (PAAG)-coated glass plates as solid support and, thus, combines the convenience of “on-membrane” staining with the sensitivity and ease of documentation of “in-gel” staining (and, therefore, is especially suited for standard lab gel documentation systems). The PAAG plates assay is compatible with all dyes for in-gel protein staining. Depending on the sensitivity of the staining protocol, the assay can be used in macro-, micro-, and nano-assay formats. We also describe a low-cost two-component colloidal Coomassie brilliant blue G-250 (CBB G-250) staining protocol for fast quantitative visualization of proteins spotted on a PAAG plate (the detection limit is up to 2ng of proteins even when using a Nikon CoolPix digital camera and white light transilluminator instead of a gel scanner). The suggested colloidal CBB G-250 protocol could also be used for visualizing nano-amounts of proteins in polyacrylamide gels. The PAAG plate assay could be useful for proteomic applications and, in general, for all cases where a fast, sensitive, and easily documentable cost-effective solid-phase protein assay is required.

改変型CBBを用いた新規Clear Native電気泳動法

改変型CBBを用いた新規Clear Native電気泳動法

Systematic comparison of technical details in CBB methods and development of a sensitive GAP stain for comparative proteomic analysis

Considering the importance of CBB staining in visualizing proteins in 2-DE gels, any improvement in the existing protocols with high sensitivity and good MS compatibility is of significant importance. In this study, we systematically evaluated the effects of different staining parameters on CBB methods by 1-DE and 2-DE, and demonstrated that G-250 was more suitable for visualizing low-abundant proteins as well as generating more spots than R-250, whereas R-250 had a superior capability for quick staining of high-abundant proteins. The staining produced by mixing G-250 and R-250 in different ratios showed similar sensitivity. Compared with acetic acid, phosphoric acid produced more protein spots. Ammonium-based stain demonstrated a superior sensitivity than the aluminum-based one. Based on these findings, a new protocol using CBB G-250, ammonium sulfate and phosphoric acid (GAP) was developed by incorporating the fixation, sensitization and staining procedures together. The comparison of GAP with other methods revealed that GAP generated more protein spots and had wider applications. The identification of 11 proteins demonstrated that GAP was not only compatible with MS but also obviously reduced in vitro protein modification, and thus could be a preferable protocol in the future proteomic analysis.

Increasing manganese peroxidase production and biodecolorization of triphenylmethane dyes by novel fungal consortium

A fungal consortium-SR consisting of Trametes sp. SQ01 and Chaetomium sp. R01 was developed for decolorizing three kinds of triphenylmethane dyes, which were decolorized by individual fungi with low efficiencies. The fungal consortium-SR produced 1.3Uml−1 of manganese peroxidase, 5.5 times higher than that produced by the monoculture of Trametes sp. SQ01, and decolorized Crystal Violet, Coomassie Brilliant Blue G250 (CBB G250) and Cresol Red. The fungal consortium-SR had a decolorization rate of 63–96%, much higher than that of the monoculture of strain SQ01 (38–72%). In consortium-SR, the higher efficiencies of decolorization of Crystal Violet and CBB G250 were obtained when they added to the culture after 4d of mixed cultivation rather than at the beginning of cultivation. Cresol Red was the exception. It is suggested that the consortium-SR has great potential for decolorizing triphenylmethane dyes.

Comparing standard and microwave assisted staining protocols for SDS-PAGE of glycoproteins followed by subsequent PMF with MALDI MS

The detection of glycoproteins on SDS-PAGE gels is a very challenging task as glycan moieties can inhibit the protein-dye interaction or protein-silver reaction slowing down or even completely preventing the staining process. Additionally the applied staining procedure can influence the total number of detected peptides after in-gel digestion. Three in SDS-PAGE commonly used staining procedures (silver nitrate, CBB R250 and colloidal CBB G250) were evaluated in terms of duration, sensitivity and obtainable sequence coverage after PMF. The staining procedures were performed with and without the assistance of microwave irradiation. Microwave treatment resulted in comparable band intensities and sensitivities as obtained by the original staining protocols (limit of detection for microwave assisted silver staining: 50 ng), but staining duration was significantly reduced, to 30 min for silver nitrate and to 1.5 h for CBB and cCBB staining method. PMF analysis by MALDI mass spectrometry was not affected by the microwave treatment. It was found that the total number of detected tryptic peptides has increased when applying microwave irradiation during the staining process (average sequence coverage 31-56% and 76% for Avidin).

Up-regulation of Nicotinic Receptors by Nicotine Varies with Receptor Subtype

Recent evidence suggests that in addition to alpha4beta2 and alpha3-containing nicotinic receptors, alpha6-containing receptors are present in midbrain dopaminergic neurons and involved in the nicotine reward pathway. Using heterologous expression, we found that alpha6beta2, like alpha3beta2 and alpha4beta2 receptors, formed high affinity epibatidine binding complexes that are pentameric, trafficked to the cell surface, and produced acetylcholine-evoked currents. Chronic nicotine exposure up-regulated alpha6beta2 receptors with differences in up-regulation time course and concentration dependence compared with alpha4beta2 receptors, the predominant high affinity nicotine binding site in brain. The alpha6beta2 receptor up-regulation required higher nicotine concentrations than for alpha4beta2 but lower than for alpha3beta2 receptors. The alpha6beta2 up-regulation occurred 10-fold faster than for alpha4beta2 and slightly faster than for alpha3beta2. Our data suggest that nicotinic receptor up-regulation is subtype-specific such that alpha6-containing receptors up-regulate in response to transient, high nicotine exposures, whereas sustained, low nicotine exposures up-regulate alpha4beta2 receptors.

A coupled reagent of o-phthalaldehyde and sulfanilic acid for protein detection based on the measurements of light scattering signals with a common spectrofluorometer

A rapid and sensitive method for the determination of proteins is proposed with a coupled reagent of o-phthalaldehyde and sulfanilic acid by measuring the light scattering (LS) signals with a common spectrofluorometer. Mechanism investigations showed that o-phthalaldehyde couples at first with sulfanilic acid with fast speed and forms a new synthesized Schiff base dye, which then interacts with protein rapidly on acidic condition, resulting in greatly enhanced LS signals with the maximum peak located at 344 nm. Based on the linear relationship between enhanced LS intensities and concentrations of proteins, a novel assay of HSA and BSA is established in the linear range of 0.1–25.0 μg ml −1 with the limits of detection (3 σ) being 13 ng ml −1 depending on the concentration of the reagent. Results for sample detections of our method were consistent with the documented spectrophotometric method with CBB G250 assay.

Studies on the Spectra of 4,5-Dibromofluorescein with Proteins and its Analytical Application

4,5-Dibromofluorescein(R) binding to proteins causes a decrease in the fluorescence maximum of R at 530 nm; the intensity of fluorescence quenching is directly proportional to protein content, Based on this, a new fast and simple fluorescence quenching method for the determination of proteins was developed. Under experimental conditions, the linear range of this assay is 0~15 mg/L and the detection limit is 40 µg/L. The method has been applied to the analysis of human serum samples and gave values close to those obtained by the Coomassie brilliant blue G-250 (CBB G-250) method, which indicated that the method is not only high in sensitivity but also reliable.

Voltammetric albumin quantification based on its interaction with carminic acid

In a weak acidic Britton-Robinson (B-R) buffer of pH 4.0, human serum albumin (HSA) can react rapidly with carminic acid (CAA) to form a supramolecular complex. Microelectrostatic fields in the molecular structure of HSA cause the electrostatic binding reaction of the positively charged CAA with the negatively charged HSA. CAA has a sensitive, second order derivative linear sweep voltammetric reductive peak at - 0.54 V (vs. SCE) on a dropping mercury electrode (DME). After the addition of HSA to a CAA solution, the reductive peak current of CAA decreased without a shift of the peak potential. Based on the decrease of the peak current, a sensitive voltammetric method for the determination of microamounts of the protein is proposed. The conditions for the binding reaction and the voltammetric detection were optimized. Under the optimal conditions, the decrease of the voltammetric detection were optimized. Under the optimal conditions, the decrease of the voltammetric peak current was in proportion to the quantity of HSA in the range of 2.0 to 70.0 mg l-1, with a linear regression equation ?Ip"(nA)=11.74 C (mg l-1)-12.33 and a detection limit of 2.0 mg l-1 (3?). The method was further applied to the detection of different kinds of proteins, such as bovine serum albumin (BSA), oval albunim (OVA), lipase, etc. The established method was further applied to the determination of the albumin content in human serum samples and the results obtained by this method were in good agreement with the traditional Coomassie Brilliant Blue G-250 (CBB G-250) spectrophotometric method. The binding mechanism is discussed and the binding ratio was calculated from the voltammetric data.

Fluorescent microscopic determination of proteins in human serum with the self-ordered ring of nuclear fast red formed on the solid support of glass slides

A self-ordered ring (SOR) technique based on the assembly of fluorescent molecules on the solid support of glass slides is presented for the detection of trace amount of proteins by using fluorescence microscope. At pH 6.62 and with the aid of poly(vinyl alcohol)-124 (PVA-124), a droplet of nuclear fast red (NFR) solution can form a fluorescent SOR on hydrophobic glass slide, and the presence of proteins can enhance its fluorescence intensity. When the volume of the droplet is 0.2 μl, linear response is observed in the range 0–100 pg, and the limit of determination is 2.44 pg for BSA and 0.91 pg for HSA (3 σ). The results of determination for three human serum samples were identical with those obtained according to the Bradford method using Coomassie Brilliant Blue (CBB G-250).

Fluorometric method for the microdetermination of protein using indigo carmine

A simple, rapid, sensitive and reproducible fluorometric method for determining microamount protein is developed. The method is based on the reaction between protein and Indigo Carmine (IC) in an aqueous solution, which results in a long-wave fluorescence emission (λ ex=690 nm, λ em=780 nm). The calibration curves of several proteins are given. Under the optimized experimental conditions, there is little interference from amino acids, some drugs, and most metal ions. In the detection of total amount of proteins in human serum, this method gives values close to those of the CBB G-250 method.

Determination of Proteins Based on Their Resonance Light Scattering Enhancement Effect on Alcian Blue 8GX

A protein assay with the determination sensitivity at nanogram levels has been developed based on measurement of enhanced resonance light scattering (RLS). In a neutral aqueous medium, the interactions of ABGX with different proteins were found to result in strongly enhanced RLS signals. With the enhanced RLS signals at 398.0 nm, proteins, including bovine serum albumin (BSA), human serum albumin (HAS), pepsin (Pep) and cellulase (Cel), can be determined with the limit of detection below 30 ng/ml. The results of determinations for artificial samples were in agreement with the desired values, and those for human serum samples were identical with those obtained according to the Bradford method using CBB G-250.

Determination of protein concentration by enhancement of the preresonance light-scattering of α,β,γ,δ-tetrakis(5-sulfothienyl)porphine

A method of protein determination with the limit of determination at nanogram levels is proposed by using a common spectrofluorometer to detect the intensity of preresonance light-scattering (PRLS). In the pH range 1.81–4.10, the interactions of α,β,γ,δ-tetrakis(5- sulfothienyl)- porphine, T(5-ST)P, with proteins were studied. It was found that the interactions result in a strongly enhanced preresonance light-scattering signal at 472.0 nm. Mechanism studies showed that the enhanced preresonance light-scattering stems from the J-aggregation of T(5-ST)P in the presence of proteins. It was found that the J-aggregation process is speedy and is scarcely affected by temperature, which supplies a precise method for the determination of proteins. Different proteins in the range 0–7 µg ml–1 can be determined with the limits of determination below 100 ng ml–1 depending on the concentration of T(5-ST)P. The results of determination for synthetic samples were in agreement with the desired values, and the ones for human serum samples were identical to those obtained according to the Bradford method using CBB G-250.

Determination of Albumin and Globulin at Nanogram Levels by a Resonance Light-Scattering Technique with α,β, γ, δ-Tetrakis(4-sulfophenyl)porphine

A method for determination of bovine serum albumin (BSA) and γ-globulin (γ-IgG) at nanogram levels is proposed by using a common spectrofluorometer to detect the intensity of resonance light-scattering. In the presence of BSA or γ-IgG at pH 1.86 and ionic strength 0.04, the aggregation of α,β,γ,δ-tetrakis(4-sulfophenyl)porphine (TPPS4) was observed. It was found to result in strong enhanced resonance light-scattering (RLS) signal with a scattering peak at 490.0 nm. But at pH 1.86 and ionic strength 0.11, only BSA can induce the aggregation of TPPS4 resulting in the strong enhanced RLS. Determination for synthetic samples by making use of the effect of ionic strength showed that if the ratio of γ-IgG in the mixture of γ-IgG and BSA is lower than 0.20, the results for the simultaneous determination of BSA and γ-IgG fractions without separation are satisfactory, but if the content of γ-IgG in the mixture is too high, the determination error is significant. However, the total content of BSA and γ-IgG in human serum can be determined with results identical to those obtained according to the Bradford method using CBB G-250.