Sodium Dodecyl Polyacrylamide Gel Electrophoresis Research Articles

Immunogenicity of different nanoparticle adjuvants containing recombinant RBD coronavirus antigen in animal model.

Ongoing mutations of SARS-CoV-2 present challenges for vaccine development, promising renewed global efforts to create more effective vaccines against coronavirus disease (COVID-19). One approach is to target highly immunogenic viral proteins, such as the spike receptor binding domain (RBD), which can stimulate the production of potent neutralizing antibodies. This study aimed to design and test a subunit vaccine candidate based on the RBD. Bioinformatics analysis identified antigenic regions of the RBD for recombinant protein design. In silico analysis identified the RBD region as a feasible target for designing a recombinant vaccine. Bioinformatics tools predicted the stability and antigenicity of epitopes, and a 3D model of the RBD-angiotensin-converting enzyme 2 complex was constructed using molecular docking and codon optimization. The resulting construct was cloned into the pET-28a (+) vector and successfully expressed in Escherichia coli BL21DE3. As evidenced by sodium dodecyl-polyacrylamide gel electrophoresis and Western blotting analyses, the affinity purification of RBD antigens produced high-quality products. Mice were immunized with the RBD antigen alone or combined with aluminum hydroxide (AlOH), calcium phosphate (CaP), or zinc oxide (ZnO) nanoparticles (NPs) as adjuvants. Enzyme-linked immunosorbent assay assays were used to evaluate immune responses in mice. In-silico analysis confirmed the stability and antigenicity of the designed protein structure. RBD with CaP NPs generated the highest immunoglobulin G titer compared to AlOH and ZnO after three doses, indicating its effectiveness as a vaccine platform. In conclusion, the recombinant RBD antigen administered with CaP adjuvant NPs induces potent humoral immunity in mice, supporting further vaccine development. These results contribute to ongoing efforts to develop more effective COVID-19 vaccines.

Proteomic alterations in various plant tissues of maize under induced chromium stress

Heavy metal contamination is becoming a major cause of pollution in the environment, impacting humans, animals, and plants directly. Because of its widespread use in the tanning industry, chromium (Cr) regarded as a highly dangerous environmental toxin. The goal of this study was to investigate growth and proteins changes in different plant tissues (leaves, shoots, and seeds) of two maize cultivars (NMH-360 and DKC 61-42) under the stress of Cr (0, 50, and 150 ppm Cr). Sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) was used to observe plant proteome modification in response to Cr stress. Results revealed that at 150 ppm, both maize cultivars showed a decrease in plant growth attributes. Furthermore, it was noticed that the plant proteome changed in response to Cr stress in leaf and shoot tissues. A few proteins were up-regulated (70 kDa in C1 variety), while others were down-regulated (154, 140, 115, 80, 70 and 53 kDa in C1 cultivar shoots while 154, 65, 60, 17 kDa bands in shoots of C2 cultivar). Some protein bands were induced at 50 ppm, some at 150 ppm, and yet others at both concentrations. The findings of this study could aid in the selection of Cr-tolerant plant cultivars as well as the design of new protein biomarkers that can be utilized as a monitoring tool in heavy metal stress responses.

Enzymatic modification of Fish Gelatin and Beet Pectin using Horseradish peroxidase

The Fish Gelatin (FG), a good alternative for unhealthy and limited socio-cultural mammalian gelatin appears to possess endogenous structural limitations. The goal of this work was to use enzymatic crosslinking to modify cold-water Fish Gelatin (FG) with Beet Pectin. Reaction conditions were optimized by a single factorial experiment and covalent crosslinking was measured by ultraviolet (UV)-Vis spectroscopy at 340 nm to indicate Horseradish Peroxidase (HRP) catalyzes Beet Pectin (BP). At 50 °C for 4 h, the highest weight ratio of heterologous adducts between FG-BP was 1:3, with HRP and Hydrogen peroxide (H2O2) of 2 µg/mL and 0.067%, (v/v), respectively. Intermolecular cross-linking was found between treated samples using ATR-FTIR and Sodium Dodecyl Sulphur and Polyacrylamide Gel Electrophoresis (SDS-PAGE). The heterologous product, control FG, and BP as well as a mixture of untreated FG-BP had a β-sheet of 41.14%, 39.65%, 39.9%, and 40.0%, respectively. The maximum reduction in elution was obtained in heterogeneous FG-BP complex. Furthermore, a schematic mechanism for Cold-water Fish Gelatin and Beet Pectin was proposed. Overall, peroxidase crosslinked BP was able to modify cold-water Fish Gelatin. The use of Horseradish peroxidase on Fish Gelatin could provide a practical way of building the FG-BP complex as a basis for understanding the FG functionalities comprehensively.

Enhancing Stability and Mucoadhesive Properties of Chitosan Nanoparticles by Surface Modification with Sodium Alginate and Polyethylene Glycol for Potential Oral Mucosa Vaccine Delivery.

Background: The present study aimed to fabricate surface-modified chitosan nanoparticles with two mucoadhesive polymers (sodium alginate and polyethylene glycol) to optimize their protein encapsulation efficiency, improve their mucoadhesion properties, and increase their stability in biological fluids. Method: Ionotropic gelation was employed to formulate chitosan nanoparticles and surface modification was performed at five different concentrations (0.05, 0.1, 0.2, 0.3, 0.4% w/v) of sodium alginate (ALG) and polyethylene glycol (PEG), with ovalbumin (OVA) used as a model protein antigen. The functional characteristics were examined by dynamic light scattering (DLS), X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and scanning electron microscopy (SEM)/scanning transmission electron microscopy (STEM). Stability was examined in the presence of simulated gastric and intestinal fluids, while mucoadhesive properties were evaluated by in vitro mucin binding and ex vivo adhesion on pig oral mucosa tissue. The impact of the formulation and dissolution process on the OVA structure was investigated by sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) and circular dichroism (CD). Results: The nanoparticles showed a uniform spherical morphology with a maximum protein encapsulation efficiency of 81%, size after OVA loading of between 200 and 400 nm and zeta potential from 10 to 29 mV. An in vitro drug release study suggested successful nanoparticle surface modification by ALG and PEG, showing gastric fluid stability (4 h) and a 96 h sustained OVA release in intestinal fluid, with the nanoparticles maintaining their conformational stability (SDS-PAGE and CD analyses) after release in the intestinal fluid. An in vitro mucin binding study indicated a significant increase in mucin binding from 41 to 63% in ALG-modified nanoparticles and a 27–49% increase in PEG-modified nanoparticles. The ex vivo mucoadhesion showed that the powdered particles adhered to the pig oral mucosa. Conclusion: The ALG and PEG surface modification of chitosan nanoparticles improved the particle stability in both simulated gastric and intestinal fluids and improved the mucoadhesive properties, therefore constituting a potential nanocarrier platform for mucosal protein vaccine delivery.

Fingerprinting of Rice (Oryza sativa) Landraces from Three Geopolitical Zones in Ebonyi State Nigeria using SDS-page

The genetic variability among ten (10) accessions of Rice (Oryza sativa) from three (3) Geopolitical zones (Ikwo, Afikpo and Abakaliki) in Ebonyi State Nigeria were assessed using sodium dodecyl polyacrylamide gel electrophoresis (SDS - PAGE). There were variations in the banding patterns of the accessions and polymorphism was also observed. The banding patterns of the 10 accessions were examined and photographed. The number of reproducible bands was recorded by observation of gels and photographs. The molecular weights of the protein bands were deduced using molecular weights of the pro-mega standard and dendrogram was also constructed in order to place different clusters and groups of the 10 accessions in the right places for easy identification. Twelve polypeptide bands were obtained ranging from 10KDa to 245KDa. The Unweight Pair Group Method with Mean Algorithm (UPGMA) dendrogram grouped the ten accessions into two clusters and five groups. Some accessions appeared in the same group, while others were observed to be in different groups. Accessions ( 3, 5, 6 and 7) neither show any atom of similarities with each other nor with the remaining accessions in terms of banding patterns and number of bands (they are significantly different) so they were placed on Group 5. This significant difference for most of the characters is an indication of wide genetic variability among the collections, which could be exploited in selection and breeding for improved varieties.

On‐Chip Electrical Monitoring of Real‐Time “Soft” and “Hard” Protein Corona Formation on Carbon Nanoparticles

AbstractWhen nanoparticles encounter a biological fluid, proteins, lipids, and other biomolecules adsorb on the nanoscale surface consequently leading to the evolution of a protein shell or “corona.” The corona formed is dynamic in nature and depends on the “synthetic identity” of the NPs, ultimately affecting their biological response. In this paper, an integrated microfluidic platform coupled with an electrical resistance measurement setup is developed to monitor and investigate the real‐time formation of a biomolecular corona of carbon nanoparticles. “Soft” and “hard” corona formation stages are effectively discriminated based on their nanoscale surface chemistries when combined with a time‐frequency tool known as wavelet transform and machine‐learning techniques. Additionally, the corona and its composition are studied using different techniques such as dynamic light scattering, nanoparticle tracking analysis, zeta potential, excitation–emission profiles, 1D sodium dodecyl polyacrylamide gel electrophoresis and subsequently, liquid chromatography‐mass spectrometry analysis. The dynamic setup can eventually be used as a valuable tool for screening of any nanoparticles formulations with distinct surface chemistries for the purpose of reduced protein adsorption/weaker protein corona formation and consequently enhance the success of targeted drug delivery.

Proteomic analysis of the phytogenic fungus Sclerotinia sclerotiorum

Sclerotinia sclerotiorum is a necrotrophic phytopathogen that has been the subject of several scientific research efforts. Despite the numerous research efforts its proteome remains understudied. This study aimed to identify proteins produced by S. sclerotiorum, thereby increasing the current proteomic knowledge base. Total proteins were extracted from mycelia scraped from five-day old cultures of S. sclerotiorum. The extracted proteins were separated by sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) and were annotated using the AB Sciex TripleToF 6600 mass spectrometer. Exactly 1471 proteins were reproducibly present in all three replicates. All proteins detected were classified based on their molecular and biological functions. To the knowledge of the authors, this is the most comprehensive proteomic study on S. sclerotiorum (judging by the high number of proteins identified).

Value of Seed Protein Profile in the Taxonomy of cultivars of Capsicum in Nigeria

Over a decade, the taxonomy of the genus Capsicum in Nigeria has remained largely unrevised, unclassified and unidentified. As such, there is a dearth of information on the proper identification of Capsicum spp and relatives found in the country. The aim of this study was to re-examine the taxonomic status of the Capsicum in Nigeria in order to establish genetic diversity between them for proper identification and classification. Sodium dodecyl polyacrylamide gel electrophoresis of total seed protein was performed on five varieties of Nigerian Capsicum spp., following standard procedures. Six protein bands were observed across the five cultivars of Capsicum, of which 12-14 Kda was the only polymorphic band. Only C. fructescens var. ijosi and C. fructescens var. sombo were unique for manifesting 20-24 and 15-16 Kda bands respectively. Dendrogram of analysis obtained resolved the taxa into two distinct groups. In the first group were cultivars of C. fructescens var. ijosi and sombo while in the second group were C. chinense, which was distinctly separated from C. fructescens var. bawa and C. annum. Artificial dichotomous key was constructed for the identification of members of the genus Capsicum available in Nigeria based on the protein profiles of their seeds.
 Keywords: Capsicum, seed protein, electrophoresis, identification, diversity.

Mass spectrometry of in-gel digests reveals differences in amino acid sequences of high-molecular-weight glutenin subunits in spelt and emmer compared to common wheat.

High-molecular-weight glutenin subunits (HMW-GS) play an important role for the baking quality of wheat. The ancient wheats emmer and spelt differ in their HMW-GS pattern compared to modern common wheat and this might be one reason for their comparatively poor baking quality. The aim of this study was to elucidate similarities and differences in the amino acid sequences of two 1Bx HMW-GS of common wheat, spelt and emmer. First, the sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) system was optimized to separate common wheat, spelt and emmer Bx6 and Bx7 from other HMW-GS (e.g., 1Ax and 1By) in high concentrations. The in-gel digests of the Bx6 and Bx7 bands were analyzed by untargeted LC-MS/MS experiments revealing different UniProtKB accessions in spelt and emmer compared to common wheat. The HMW-GS Bx6 and Bx7, respectively, of emmer and spelt showed differences in the amino acid sequences compared to those of common wheat. The identities of the peptide variations were confirmed by targeted LC-MS/MS. These peptides can be used to differentiate between Bx6 and Bx7 of spelt and emmer and Bx6 and Bx7 of common wheat. The findings should help to increase the reliability and curation status of wheat protein databases and to understand the effects of protein structure on the functional properties. Graphical abstract.

Interaction of Porcine Myofibrillar Proteins and Various Gelatins: Impacts on Gel Properties.

The objectives of this study were to determine the interaction between porcine myofibrillar proteins and various gelatins (bovine hide, porcine skin, fish skin, and duck skin gelatins) and their impacts on gel properties of porcine myofibrillar proteins. Porcine myofibrillar protein was isolated from pork loin muscle (M. longissimus dorsi thoracis et lumborum). Control was prepared with only myofibrillar protein (60 mg/mL), and gelatin treatments were formulated with myofibrillar protein and each gelatin (9:1) at the same protein concentration. The myofibrillar protein-gelatin mixtures were heated from 10°C to 75°C (2°C/min). Little to no impacts of gelatin addition on pH value and color characteristics of heat-induced myofibrillar protein gels were observed (p>0.05). The addition of gelatin slightly decreased cooking yield of heat-induced myofibrillar protein gels, but the gels showed lower centrifugal weight loss compared to control (p<0.05). The addition of gelatin significantly decreased hardness, cohesiveness, gumminess, and chewiness of heat-induced myofibrillar gels. Further, sodium dodecyl poly-acrylamide gel electrophoresis (SDS-PAGE) showed no interaction between myofibrillar proteins and gelatin under non-thermal conditions. Only a slight change in the endothermic peak (probably myosin) of myofibrillar protein-gelatin mixtures was found. The results of this study show that the addition of gelatin attenuated the water-holding capacity and textural properties of heat-induced myofibrillar protein gel. Thus, it could be suggested that well-known positive impacts of gelatin on quality characteristics of processed meat products may be largely affected by the functional properties of gelatin per se, rather than its interaction with myofibrillar proteins.

Quantity and Functionality of Protein Fractions Isolated from 3 Ecotypes of Indigenous Chicken in Kenya

&lt;p&gt;&lt;em&gt;The aim of this study was to evaluate the effect of the cluster ecotype and the part of chicken on nutritional composition, and functionality of sarcoplasmic and myofibrillar proteins that are most relevant to the technological features of chicken meat. Over 50 chickens from each ecotype cluster purchased, slaughtered and the meat stored under refrigeration at -20&lt;sup&gt;o&lt;/sup&gt;C and later on transferred in cooler box on ice and flown to South Africa, at the Durban University of Technology. Protein fractions were extracted with a cocktail of Sodium Chloride buffer (50mM NaCl, 50mM Tris HCl; 75mM DTT and 1mM EDTA at pH 7) and quantified by Bradford method. One dimensional Sodium Dodecyl Polyacrylamide Gel Electrophoresis (SDS PAGE) was applied to separate protein fractions. Emulsifying capacity, emulsifying stability, solubility, and in vitro digestibility were determined on the total protein isolates. Significant differences in band expressions were recorded for the myofibrillar and the sarcoplasmic proteins. The three ecotypes had high quality proteins with all the limiting and essential amino acids at concentrations higher than FAO/WHO recommended daily allowance for adults and children. Distinct protein bands at larger molecular weight proteins &amp;gt;100 kDa, corresponding to Myosin Heavy Chain, medium fractions 75 kDa and 45 kDa and even lower molecular weight fraction &amp;lt;25 kDa were present in the chicken breast and the thighs. It concludes that Indigenous chicken protein isolates’ nutritional and functional properties are affected by part of chicken and ecotype clusters. &lt;/em&gt;&lt;/p&gt;

Assessment of Antiglycation Effects of Some Plants Used in Hypolipidaemic Formulations or as Spices

Aims: Diabetes mellitus is characterized by hyperglycemia, which leads to acceleration of protein glycation. Some glycation products form inter molecular cross-links, contributing to chronic diabetic complications. Determination of protein glycation inhibitory potential of medicinal plants may offer safe therapeutic avenues in preventing diabetic complications. The aim of this study was to analyze the antiglycation effects of nine plants which included spices and some medicinal plants used in hypolipidaemic formulations. Study Design: Experimental. Place and Duration of Study: Department of Biochemistry, Faculty of Medicine, University of Peradeniya, Sri Lanka between March 2014 and March 2015. Methodology: Crude methanol extracts of nine, dried plant parts namely, Brassica juncea (BJ) seed, Coscinium fenestratum (CF) root, Cuminum cyminum (CC) seed, Cyperus rotundus (CR) root, Foeniculum vulgare (FV) seed, Picrorhiza kurroa (PK) root, Acorus calamus (AC) rhizome, Trachyspermum roxburghianum (TR) leaf and Trigonella foenum-graecum (TF) seed and standard inhibitor aminoguanidine (AG) (1 mg/mL) were used. Inhibitory effects of plant extracts (0.5/ 2/ 5 mg/mL) on fructosamine formation, glycation and glycation induced cross-linking were assessed. Results: Fructosamine formation was significantly inhibited (p<0.001) in the presence of CF, CR, PK and AC (2 mg/ ml). Antiglycation effects of these four plants were demonstrated when the extent of glycation was assessed using polyacrylamide gel electrophoresis. Protein cross-linking inhibitory effects of AC, CF, CR and PK extracts (2 mg/ ml) were revealed using sodium dodecyl polyacrylamide gel electrophoresis. At 0.5 mg/ ml, this inhibitory effect remained only in CR and PK. When 5 mg/ ml was used, BJ showed inhibitory effects while CC, TR and TF without an effect. Accordingly, the highest inhibitory effects were seen in CR and PK. Conclusion: Antiglycation effects of root extracts of AC, CF, CR and PK were demonstrated with better effects in CR and PK.

Striking Effects of Storage Buffers on Apparent Half-Lives of the Activity of Pseudomonas aeruginosa Arylsulfatase.

To obtain the label enzyme for enzyme-linked-immunoabsorbent-assay of two components each time in one well with conventional microplate readers, molecular engineering of Pseudomonas aeruginosa arylsulfatase (PAAS) is needed. To compare thermostability of PAAS/mutants of limited purity, effects of buffers on the half-activity time (t 0.5) at 37°C were tested. At pH 7.4, PAAS showed non-exponential decreases of activity, with the apparent t 0.5 of ~6.0days in 50mM HEPES, but ~42days in 10mM sodium borate with >85% activity after 15days; protein concentrations in both buffers decreased at slower rates after there were significant decreases of activities. Additionally, the apparent t 0.5 of PAAS was ~14days in 50mM Tris-HCl, and ~21days in 10mM sodium phosphate. By sodium dodecyl-polyacrylamide gel electrophoresis, the purified PAAS gave single polypeptide; after storage for 14days at 37°C, there were many soluble and insoluble fragmented polypeptides in the HEPES buffer, but just one principal insoluble while negligible soluble fragmented polypeptides in the borate buffer. Of tested mutants in the neutral borate buffer, rates for activity decreases and polypeptide degradation were slower than in the HEPES buffer. Hence, dilute neutral borate buffers were favorable for examining thermostability of PAAS/mutants.

Potential Protease Inhibitor Isoforms from Pigeonpea againstHelicoverpa armigeraGut Proteases

Ten pigeon pea genotypes (four moderately resistant, three intermediate, two moderately susceptible and one unknown status) were evaluated for their efficacy against bovine trypsin and Helicoverpa armigera (H. armigera) gut proteases. The trypsin inhibitor content in pigeon pea genotypes varied from 147.75 (TIU/g) (AL 201) to 177.85 TIU/g (AL 1495). Sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) of seed extracts revealed different proteins of molecular weight ranging from 15.6 kDa to 98.5 kDa. Using gelatin SDS-PAGE, six different trypsin inhibitors with bovine trypsin were identified and four of them with relative mobility (Rm) 0.39, 0.78, 0.84 and 0.93 were present in all the ten genotypes. Five H. armigera protease inhibitors (PIs) were detected using the gelatin SDS-PAGE. Four of them also inhibited the bovine trypsin. One (35.3 kDa) was specific only for H. armigera gut proteases. Two protease inhibitors (15.6 kDa and 20.1 kDa) were specific for bovine trypsin. However, it appears that it is the unspecific PI with Rm 0.39 (51.5 kDa) and 0.93 (16.6 kDa) which mainly determine the resistance towards H. armigera in pigeonpea.

Protein Profile Study of Some Nigerian Sesame (Sesamum indicum L.) Accessions

This study was carried out to investigate the genetic diversity among 23 sesame (Sesamum indicum L.) accessions obtained from different agro-ecological localities from 10 different states across 4 geopolitical zones in Nigeria using evidence from Sodium Dodecyl Polyacrylamide Gel Electrophoresis (SDS-PAGE). Total seed protein of the studied plants resolved on 12% SDS-PAGE showed variations in numbers and intensity of bands among the different sesame accessions. Thirteen (13) major bands were recorded in this study. Lack of unique band and presence of common band (band 7) among the 23 studied sesame accessions indicate some levels of genetic affinity and evidence of common evolutionary origin of the sesame genotypes. This band can therefore be tagged as species specific band for discriminating Sesamum indicum. Cluster analysis grouped the 23 sesame genotypes into two clusters with similarity coefficient ranging from 0.42 to 0.96 which indicates existence of genetic diversity; therefore there is ample opportunity for improving the 23 sesame genotypes. Variations in protein bands observed among the 23 studied plants could be attributed to genomic changes taken place during species diversification. It can be concluded that genetic diversity existed among Nigerian sesame for the improvement of characters of interest. Accessions 9 (YOL), 15(OTT), 22 (OFF) and 23 (JAL) are therefore recommended for used in future breeding programs for the development of improved sesame varieties.Int J Appl Sci Biotechnol, Vol 3(2): 322-329 DOI: http://dx.doi.org/10.3126/ijasbt.v3i2.12734

Analysis of glycation induced protein cross-linking inhibitory effects of some antidiabetic plants and spices.

BackgroundProtein cross-linking which occurs towards the latter part of protein glycation is implicated in the development of chronic diabetic complications. Glycation induced protein cross-linking inhibitory effects of nine antidiabetic plants and three spices were evaluated in this study using a novel, simple, electrophoresis based method.MethodsMethanol extracts of thirteen plants including nine antidiabetic plants and three spices were used. Lysozyme and fructose were incubated at 37 °C in the presence or absence of different concentrations of plant extracts up to 31 days. Standard glycation inhibitor aminoguanidine and other appropriate controls were included. A recently established sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) method was used to detect the products of protein cross-linking in the incubation mixtures.ResultsHigh molecular weight protein products representing the dimer, trimer and tetramer of lysozyme were detected in the presence of fructose. Among the nine antidiabetic plants, seven showed glycation induced protein cross-linking inhibitory effects namely Ficus racemosa (FR) stem bark, Gymnema sylvestre (GS) leaves, Musa paradisiaca (MP) yam, Phyllanthus debilis (PD) whole plant, Phyllanthus emblica (PE) fruit, Pterocarpus marsupium (PM) latex and Tinospora cordifolia (TC) leaves. Inhibition observed with Coccinia grandis (CG) leaves and Strychnos potatorum (SP) seeds were much low. Leaves of Gymnema lactiferum (GL), the plant without known antidiabetic effects showed the lowest inhibition. All three spices namely Coriandrum sativum (CS) seeds, Cinnamomum zeylanicum (CZ) bark and Syzygium aromaticum (SA) flower buds showed cross-link inhibitory effects with higher effects in CS and SA. PD, PE, PM, CS and SA showed almost complete inhibition on the formation of cross-linking with 25 μg/ml extracts.ConclusionsMethanol extracts of PD, PE, PM, CS and SA have shown promising inhibitory effects on glycation induced protein cross-linking.Electronic supplementary materialThe online version of this article (doi:10.1186/s12906-015-0689-1) contains supplementary material, which is available to authorized users.

Biosynthesis of Silver Chloride Nanoparticles Using Bacillus subtilis MTCC 3053 and Assessment of Its Antifungal Activity

The present investigation reported the synthesis of silver chloride nanoparticles using Bacillus subtilis. The adsorption of colloidal silver chloride nanoparticles showed an intense peak at the wavelength of 400 nm after 20 hrs of biomass incubation. The size of the silver nanoparticles ranges from 20 to 60 nm which was obtained from transmission electron microscope (TEM). The X-ray diffraction (XRD) pattern confirmed the crystalline nature of the nanoparticles. The bright circular spots of selected diffraction area (SAED) pattern also confirmed the good crystalline nature of the silver chloride nanoparticles with high magnification of TEM images. The presence of nitrate reductase enzyme in the cellular membrane of B. subtilis was confirmed by sodium dodecyl (SDS) polyacrylamide gel electrophoresis and it was found that the molecular weight is 37 kDa. The possible functional groups of the reductase enzyme in B. subtilis were identified by Fourier transform infrared spectroscopy (FTIR). Finally, antifungal activity of silver chloride nanoparticle was examined against Candida albicans, Aspergillus niger, and Aspergillus flavus. We conclude that the synthesis of silver chloride nanoparticles using microorganisms is more economical and simple. The antifungal property of silver chloride nanoparticles will play a beneficial role in biomedical nanotechnology.

Performing Isoelectric Focusing and Simultaneous Fractionation of Proteins on A Rotary Valve Followed by Sodium Dodecyl–Polyacrylamide Gel Electrophoresis

In this technical note, we design and fabricate a novel rotary valve and demonstrate its feasibility for performing isoelectric focusing and simultaneous fractionation of proteins, followed by sodium dodecyl-polyacrylamide gel electrophoresis. The valve has two positions. In one position, the valve routes a series of capillary loops together into a single capillary tube where capillary isoelectric focusing (CIEF) is performed. By switching the valve to another position, the CIEF-resolved proteins in all capillary loops are isolated simultaneously, and samples in the loops are removed and collected in vials. After the collected samples are briefly processed, they are separated via sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE, the second-D separation) on either a capillary gel electrophoresis instrument or a slab-gel system. The detailed valve configuration is illustrated, and the experimental conditions and operation protocols are discussed.

Purification and properties of S-hydroxymethylglutathione dehydrogenase of Paecilomyces variotii no. 5, a formaldehyde-degrading fungus.

S-hydroxymethylglutathione dehydrogenase from Paecilomyces variotii No. 5 strain (NBRC 109023), isolated as a formaldehyde-degrading fungus, was purified by a procedure that included ammonium sulfate precipitation, DEAE-Sepharose and hydroxyapatite chromatography and isoelectrofocusing. Approximately 122-fold purification was achieved with a yield of 10.5%. The enzyme preparation was homogeneous as judged by sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE). The molecular mass of the purified enzyme was estimated to be 49 kDa by SDS-PAGE and gel filtration, suggesting that it is a monomer. Enzyme activity was optimal at pH 8.0 and was stable in the range of pH 7.0–10. The optimum temperature for activity was 40°C and the enzyme was stable up to 40°C. The isoelectric point was pH 5.8. Substrate specificity was very high for formaldehyde. Besides formaldehyde, the only aldehyde or alcohol tested that served as a substrate was pyruvaldehyde. Enzyme activity was enhanced by several divalent cations such as Mn2+ (179%), Ba2+ (132%), and Ca2+ (112%) but was completely inhibited by Ni2+, Fe3+, Hg2+, p-chloromercuribenzoate (PCMB) and cuprizone. Inactivation of the enzyme by sulfhydryl reagents (Hg2+ and PCMB) indicated that the sulfhydryl group of the enzyme is essential for catalytic activity.

Immobilization of albumin on magnetite nanoparticles

The magnetite (Fe 3O 4) nanoparticles were prepared by the co-precipitation of ferrous and ferric salts with NH 4OH, and then modified with 3-aminopropyltriethoxysilane (APTES) by silanization reaction and subsequent reaction with glutaraldehyde (GA) to obtain functional groups on their surface. The influence of different terminated groups on protein binding was studied with bare and modified magnetite nanoparticles. Amine terminated magnetite nanoparticles were shown the highest binding ability for immobilization process compared to Fe 3O 4 NPs and GA bonded NPs. This binding ability was shown by using sodium dodecyl polyacrylamide gel electrophoresis technique (SDS-PAGE). Albumin attached magnetite nanoparticles were also examined by Scanning Electron Microscopy (SEM).