Mixture Of Bovine Serum Albumin Research Articles

Electrophoretic Transport of Biomolecules through Carbon Nanotube Membranes

Electrophoretic transport of proteins across electrochemically oxidized multi-walled carbon nanotube (MWCNT) membranes has been investigated. A small charged protein, lysozyme, was successfully pumped across MWCNT membranes by an electric field while rejecting larger bovine serum albumin (BSA). Transport of lysozome was reduced by a factor of about 30 in comparison to bulk mobility and consistent with the prediction for hindered transport. Mobilities between 0.33 and 1.4 × 10(-9) m(2) V(-1) s(-1) were observed and are approximately 10-fold faster than comparable ordered nanoporous membranes and consistent with continuum models. For mixtures of BSA and lysozyme, complete rejection of BSA is seen with electrophoretic separations.

Adsorption of humic acid onto ultrafiltration membranes in the presence of protein and metal ions

In this research, adsorption of humic acid (HA) from HA–BSA (bovine serum albumin) mixtures onto ultrafiltration membranes was elucidated in the presence of various metal ions. Three equilibrium adsorption isotherms i.e. Langmuir, Freundlich and Redlich-Peterson were tested to fit to the experimental sorption data. The Redlich-Peterson isotherm was found to well represent the measured adsorption data for all studied cases based on the higher coefficient of determination ( r 2). Influence of some factors on adsorption mechanism of humic acid including BSA addition, ionic strength, ionic charge and size of hydrated ion were investigated. Adsorption kinetic can be interpreted as two distinct regions (during filtration time) based on the intensity and relative stability of intermolecular interactions i.e. transient and steady state. At transition state electrostatic interactions between solutes and HA-macromolecules are more effective than interactions between membranes and HA. At steady state region intermolecular interactions lead to establishment of electrochemical bridges between solution's components and molecular aggregates form in the solution leads to superior adsorption on the membrane compared to transient state region. An increment in ionic strength and charge of ions in the solution improves the final sorption capacity of the membranes. However this initially decreases the sorption of HA on the membranes at transient state. Cation with lower charge density (e.g. Ba + 2 ) strongly attracts negatively charged humic acid molecules leading to higher sorption onto the membrane. There is less water molecules surrounding the cations with lower charge density. Cations with higher charge density (e.g. Mg + 2 ) attract more water molecules and therefore the cation's effective charge is shielded leading to less adsorption of HA-molecules.

Elution Behavior of Proteins in Capillary Chromatography Using an Untreated Fused-silica Capillary Tube and a Water–Hydrophilic–Hydrophobic Organic Mixture Carrier Solvent

Abstract We examined the elution behavior of isoluminol isothiocyanate (ILITC)-labeled proteins (alcohol dehydrogenase (ADH) and bovine serum albumin (BSA)) in a capillary chromatography system using an untreated fused-silica capillary tube and a water–acetonitrile–ethyl acetate mixture carrier solution. The mixture of ILITC and ILITC-labeled protein (ADH or BSA) was analyzed using this system with a chemiluminescence detector; the ILITC and the labeled protein were separated in this order with an organic solvent-rich carrier solution, while they were eluted in the reverse order with a water-rich carrier solution. In addition, the mixture of ILITC, ILITC-labeled ADH, and ILITC-labeled BSA was added to this system, and the analytes of the mixture were separated and confirmed with the individual peaks on the chromatograms.

ELISA for antiphosphatidylethanolamine antibody detection: High impact of assay buffer on results

The aim of this study was to evaluate the influence on the results of the main variables of ELISA used for the detection of antiphosphatidylethanolamine antibodies (aPE). Forty sera from patients with either autoimmune disorders including antiphospholipid syndrome (APS) or the clinical features of APS only were assayed by ELISA performed under different conditions. Variables were sources of PE (egg yolk, soybean, bovine brain or E scherichia coli), microtiter plates (plain or gamma irradiated) and buffer components-fetal calf serum (FCS), adult bovine plasma (ABP), adult bovine serum (ABS) or bovine serum albumin (BSA). aPE binding was decreased with PE from E. coli while the other tested PE gave comparable results. The influence of the type of plates was restricted to IgM isotype with slightly, but significantly higher optical densities with plain than with irradiated plates. Most importantly, the component buffer had the highest impact on the results as shown by a strong decrease of the signal by ABP or ABS. This inhibitory effect was confirmed by using mixtures of FCS or BSA with increasing concentrations of ABS. Partial delipidation of ABS resulted in a recovery of OD levels close to those obtained with FCS. This study is the first to demonstrate that aPE reactivity is dependent on the lipid concentration of the buffer component. These results highlight the need for standardization of aPE-ELISA for a better understanding of their clinical significance.

Rhodamine B as Ligand for Affinity Chromatography: Chromatographic Studies on Derivatized Beaded Cellulose

Rhodamine B (RB) post-grafted onto beaded cellulose by a curing method is used as a biomimetic ligand in dye affinity chromatography. The grafted materials obtained are qualitatively characterized by scanning electron microscope and fourier transformed infrared spectroscopy. An amount of 76.79 mmol RB/g dyed cellulose is determined by elemental analysis. The RB affinity interaction with the trypsin, alpha-chymotrypsin, and BSA (bovine serum albumin) is analyzed using different mobile phase composition. The results show a selective separation of a mixture of BSA and trypsin into two single peaks by step elution with 1.75 M, 0.5 M, and 0 M ammonium sulphate in the eluent buffer. A good reproducibility of the retention time is obtained for these proteins in the mixture with typical values of 8.0 +/- 0.2 min for BSA and 20.0 +/- 0.2 min for trypsin, showing a possible application in the purification of samples with different composition.

Gold nanoparticles: dispersibility in biological media and cell-biological effect

Spherical gold nanoparticles with a hydrodynamic diameter between 25 and 37 nm were prepared and stabilised with poly(N-vinylpyrrolidone) (PVP) or tris(sodium-m-sulfonatophenyl)phosphine (TPPTS). They were subjected to different cell culture media, e.g. pure RPMI, RPMI containing up to 10% of fetal calf serum (FCS), and RPMI containing up to 10% of bovine serum albumin (BSA), and the rate of agglomeration was studied by dynamic light scattering. In pure RPMI, a strong agglomeration was observed whereas in the RPMI–FCS and RPMI–BSA mixtures the particles remained well dispersed above 1 wt% protein concentration. The effect of PVP-stabilised gold nanoparticles on human mesenchymal stem cells (hMSC) was studied as well. No significant influence on the viability and chemotaxis was observed after incubation of hMSC with gold nanoparticles. However, gold nanoparticles induced the activation of hMSC as indicated by the release of IL-6 and IL-8.

The influence of proteins on the dispersability and cell-biological activity of silver nanoparticles

Spherical silver nanoparticles with a diameter of 50 ± 20 nm and stabilized with either poly(N-vinylpyrrolidone) (PVP) or citrate were dispersed in different cell culture media: (i) pure RPMI, (ii) RPMI containing up to 10% of bovine serum albumin (BSA), and (iii) RPMI containing up to 10% of fetal calf serum (FCS). The agglomeration behavior of the nanoparticles was studied with dynamic light scattering and optical microscopy of individually tracked single particles. Whereas strong agglomeration was observed in pure RPMI and in the RPMI–BSA mixture within a few hours, the particles remained well dispersed in RPMI–FCS. In addition, the biological effect of PVP-stabilized silver nanoparticles and of silver ions on human mesenchymal stem cells (hMSCs) was studied in pure RPMI and also in RPMI–BSA and RPMI–FCS mixtures, respectively. Both proteins considerably increased the cell viability in the presence of silver ions and as well as silver nanoparticles, indicating a binding of silver by these proteins.

A silicone-based microfluidic chip grafted with carboxyl functionalized hyperbranched polyglycerols for selective protein capture

A poly(dimethylsiloxane) (PDMS)-based functional microfluidic device containing a charged matrix of PDMS pillar arrays grafted with hyperbranched polyglycerols (HPGs) was developed. Samples of PDMS were modified with allylamine plasma to form amine groups on the surface prior to the covalent grafting of succinimdyl ester-functionalized HPGs. The anionic functionality of the PDMS channel matrices was developed by altering the number of carboxyl groups present on the HPGs. The grafting of HPGs onto PDMS plates was investigated via contact angle measurement and attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), while the grafting of the inside channel was investigated by electroosmotic flow (EOF) measurements. The charge density on grafted HPG was optimized to minimize the nonspecific protein adsorption and increase the selective capture of positively charged proteins. A proof-of-concept device was fabricated on PDMS and demonstrated that the device selectively captures positively charged protein (avidin) from a mixture of bovine serum albumin (BSA)-avidin at pH 7.4 in phosphate buffered saline (PBS). In order to increase the capture efficiency of the proteins in this PDMS-based device, pillar arrays have been fabricated within the channel. As a demonstration, the new device separated two proteins with an avidin capture efficiency of 100 ± 2.95% per 3 min from a 0.02 mg/ml protein solution (avidin:BSA wt ratio: 1:1). This new microfluidic-based device shows a great deal of promise as a tool for protein capture and analysis.

Chemical Approach for Specific Enrichment and Mass Analysis of Nitrated Peptides

The analysis and detection of 3-nitrotyrosine are biologically and clinically important because protein tyrosine nitration is known to be involved in a number of biological phenomena such as cellular signal transduction, pathogenesis of inflammatory responses, and age-related disorders. However, the main obstacles in the study are low abundance of nitrated species and lack of efficient enrichment methods. Here in, we suggest a new chemical approach to analyze nitrated peptides using mass spectrometry by incorporating specific tagging groups in the peptides through simple chemical transformations. Nitro groups on tyrosine side chains of nitrated peptides were subjected to reduction to give rise to amine which was further converted to metal-chelating motif. Mass analyses verified that Ni(2+)-NTA magnetic agarose beads selectively captured and isolated the modified peptides, i.e., nitrated peptides, by strong and specific metal chelating interactions. We further demonstrated the utility of our approach by detection of nitrated peptides in complex samples such as tryptic peptide mixtures of bovine serum albumin (BSA) and a HeLa cell lysate.

Dramatic performance improvement of weak anion-exchange membranes for chromatographic bioseparations

This contribution describes a two-step surface modification method to prepare high-capacity anion-exchange membranes for chromatographic bioseparations. In the first step, commercially available regenerated cellulose membranes were functionalized with initiator. In the second step, initiator-functionalized membranes were modified by surface-initiated atom transfer radical polymerization of 2-dimethylaminoethyl methacrylate (DMAEMA). Initiator grafting density and molecular weight of poly(DMAEMA) chains were varied independently to yield surface-modified, macroporous, weak anion-exchange membranes with exceptionally high protein binding capacities (dynamic capacities up to 130 mg/mL for bovine serum albumin) at high linear flow velocities (>350 cm/h) and low transmembrane pressure drop (<3 bar). Initiator grafting density was increased by increasing the concentration of initiator precursor in solution during the membrane initiator-functionalization reaction. Average molecular weight of poly(DMAEMA) chains was increased by increasing polymerization time at constant initiator grafting density. Dynamic protein binding capacities were found to be independent of the linear flow velocity. Dynamic capacities decreased in regular fashion with increasing ionic strength in the loading buffer, but maintained relatively high values (>35 mg/mL) even at 100 mM ionic strength. Separation performance of the newly designed membranes was evaluated by fractionating a mixture of bovine serum albumin and hemoglobin. The purities of recovered proteins were high (>97%), and the recoveries also were high (>98%).

Evaluation of hydroxyapatite microspheres made from a borate glass to separate protein mixtures

A hydroxyapatite (HA, Ca10(PO4)6(OH)2), transformed from a calcium-containing borate glass, has been investigated for its protein adsorption and chromatographic characteristics. Microspheres of the borate glass were transformed into HA by reacting them with a 0.25 M phosphate (K2HPO4) solution for 24 h at 37 °C (pH 9.0). The HA microspheres with a diameter of 45–90 μm were hand packed into a steel column (4.6 mm × 80 mm) and used to separate a binary protein mixture of bovine serum albumin (BSA) and lysozyme. HA microspheres, with a diameter <45 μm, were used for separating a protein mixture of BSA, myoglobin, and lysozyme. These microspheres had a diameter that was 20–30 times larger than commercial HA column packing spherical particles, 2–3 μm, but these microspheres had a six times larger surface area and a more uniform spherical shape. These advantages compensated for their larger size and the separation results were comparable to those commercially available HA columns in the separation of the proteins studied. These unique HA microspheres, made from microspheres of a borate glass, are considered to be useful as packing materials for protein separation in chromatography.

Synergy between Hofmeister effect and coupled water in proteins: Unusual dilational moduli of BSA at air/solution interface

This study relates interfacial interactions of bovine serum albumin (BSA) molecules in dilute solutions with its dilatational rheology. Dynamic surface tension and the associated dilational elastic modulus and viscosity for BSA and mixtures of BSA with Hofmeister electrolytes—NaCl, NaClO 4, Na 2SO 4, NaF and Na 2HPO 4 have been studied using a sinusoidal surface compression and expansion for frequencies ranging from 0.01 to 0.4 Hz. at solution/air interface. In all the BSA + electrolyte systems, both the elastic modulus and viscosity show unusually high values compared with pure BSA or pure electrolytes. In the presence of NaF and Na 2SO 4 the viscosity of protein increases almost by 50–80-fold and the corresponding elastic modulus also changes by 30–50-fold. Hydrated Hofmeister ions surely influence the measured rheological properties. In addition, the synergistic effect of the hydrated protein and the vicinal hydrated electrolytes possibly contribute to the high viscosity and elasticity due to change in dynamics of these assemblies. Thus the behavior of BSA is effected by salts in different ways, especially due to the dynamics and strength of the water molecules in the assembly.

Study of adsorption of bovine serum albumin to Langmuir–Blodgett film coated surfaces using work of adhesion as a tool

This work reports on the use of rate of change of work of adhesion (Δ W) as a tool to study adsorption of bovine serum albumin (BSA) to glass and Langmuir–Blodgett film of dihexadecyl phosphate (DHP) and dioctadecyl dimethyl ammonium bromide (DOMA) coated surfaces. Pure BSA and BSA with additives – sorbitol and urea – have been adsorbed to bare glass surfaces and DHP and DOMA coated surfaces. The results suggest that an increase in Δ W with time indicates promotion of adsorption while a decrease indicates hindered adsorption. Further adsorption of BSA was most effective on DHP coated surface compared with bare glass and DOMA coated glass. In case of mixtures of BSA with urea and sorbitol, BSA + urea showed hindered adsorption while adsorption of BSA + sorbitol was efficient for all substrates.

Biocomposite of double-walled carbon nanotube-doped alginate gel for biomaterial immobilization

An efficient support for biomaterial immobilization, double-walled carbon nanotube-doped alginate gel (DWCNT–ALG) hybrid composite, was studied in this work. DWCNTs were cut by mixed acids to get a homogeneous dispersion in an aqueous solution. Fourier transform infrared spectroscopy (FTIR) was employed to analyze the structure of shortened DWCNTs. Bovine serum albumin (BSA), the model biomaterial, was immobilized in DWCNT–ALG composites. Mixture of DWCNTs and BSA were suspended in ALG homogenously and then added into a calcium chloride solution dropwise to form biocomposite beads. Transmission and scanning electron microscope (TEM and SEM) images confirmed the adsorption of BSA on DWCNTs and the uniform distribution of DWCNTs in ALG, respectively. Water loss and BSA leakage from the BSA–DWCNT–ALG biocomposites were reduced to 46.48% and 19.28%, respectively, compared with those from BSA–ALG biocomposites. These DWCNT–ALG biocomposites showed high efficiency in BSA immobilization.

A batch incubation assay to screen plant samples and extracts for their ability to inhibit rumen protein degradation

A major objective in nutritional management of high yielding dairy cows is to reduce degradation of dietary protein by rumen microorganisms. The plant kingdom offers a huge diversity of complex compounds that may modify rumen proteolysis. To screen large sample collections and identify plants containing antiproteolytic compounds, rapid and efficient methods are required. Many assays to examine antiproteolytic effects are short term and address direct interferences with protease activities, but isolate the process of proteolysis from overall fermentation. This study aimed to develop a screening system based on a compound substrate, and adequate incubation times, to determine effects on carbohydrate fermentation and microbial growth as well. We describe a simple approach based on batch incubations with rumen fluid to monitor protein degradation over a 12 h incubation. To optimise the system, particular attention was given to the choice of the protein source in the mixed substrate. Casein, bovine serum albumin (BSA), RubisCo and soy protein were assessed with respect to solubility in the buffer medium, a well as detectability of single protein bands on polyacrylamide gels and degradation rates. The ionophore monensin was used as a positive control, due to its known efficacy, and effects were compared to those from in vitro and in vivo studies to validate results from our screening system. The rapid degradation of casein by rumen proteases impeded detection of monensin effects on proteolysis, thereby demonstrating that protein quality crucially impacts efficacy of additives in the assay. Finally, a mixture of soy protein and BSA at a total concentration of 169 g protein/kg was chosen as the standard. Reported effects of monensin on proteolysis and fibre degradation were demonstrated in the assay in a dose dependent manner and 3 μM monensin was chosen for routine application. The system has the advantage of distinguishing between substrate and microbial protein by a centrifugation step and directly monitoring disappearance of substrate protein or single protein bands. Concomitant measurement of gas production, SCFA, and proteolytic end products – ammonia and branched SCFA – provides additional information on overall fermentation as well as on amino acid deamination. The resulting assay was successfully applied for screening in the EC Framework V project, ‘Rumen-up’ to select antiproteolytic plants.

Fouling mechanisms of model polymeric substances

AbstractExtracellular polymeric substances (EPS) and soluble microbial products (SMP) have been identified as the main foulants in membrane bioreactor (MBR) operation. For a better understanding of the fouling mechanisms of these substances, filtration of model solutions with submerged membranes was carried out. Filtration was conducted following the flux‐stepping method. In this work alginate was used as a model for polysaccharide, bovine serum albumin (BSA) as a model for protein, and yeast (washed and unwashed) and bentonite were used for suspended solid contents. Fouling behaviours and rejection propensities were also assessed for one‐component and two‐component solutions and provided explanations for fouling mechanisms of carbohydrate and its interaction with protein and membrane in MBR systems. During the filtration of most foulants (except BSA), introducing the alginate increased the reversible fouling more than the irreversible one. In addition, a different behaviour was observed for the mixture of BSA and alginate. Fouling rate was also affected by the introduction of alginate. Copyright © 2007 Curtin University of Technology and John Wiley &amp; Sons, Ltd.

Adsorption and viscoelastic properties of fractionated mucin (BSM) and bovine serum albumin (BSA) studied with quartz crystal microbalance (QCM-D)

The adsorption profile and viscoelastic properties of bovine submaxillary gland mucin (BSM) and bovine serum albumin (BSA), extracted from a commercial mucin preparation, adsorbing to polystyrene surfaces has been studied using quartz crystal microbalance with dissipation monitoring (QCM-D). A significant difference in the adsorption properties of the different proteins was detected; with the BSA adsorbing in a flat rigid layer whilst the mucin adsorbed in a diffuse, highly viscoelastic layer. Subsequent addition of BSA to the preadsorbed mucin layer resulted in stiffening of the protein layer which was attributed to complexation of the mucin by BSA. In contrast, a preadsorbed layer of BSA prevented mucin adsorption altogether. Combined mixtures of mucin and BSA in well defined ratios revealed intermediate properties between the two separate protein species which varied systematically with the protein ratios. The results shed light on the synergistic effects of complexation of lower molecular weight biomolecular species with mucin. The possibility to selectively control protein uptake and tailor the physical properties of the adsorbed layer makes mucin an attractive option for application in biomaterial coatings.

High Performance Tangential Flow Filtration Using Charged Affinity Ligands

High performance tangential flow filtration is an emerging technology for protein separations. The objective of this work was to examine the use of small charged affinity ligands to control the protein charge and thus extend the potential applicability of this separation technique. Experiments were performed using binary mixtures of bovine serum albumin and ovalbumin, with Cibacron Blue added as an affinity ligand. The addition of Cibacron Blue caused a large increase in the selectivity due to the increase in BSA retention associated with the strong electrostatic repulsion between the negatively‐charged protein‐ligand complex and the negatively‐charged cellulosic membrane. Appropriate conditions for the separation were identified using total recycle experiments in a small‐scale tangential flow filtration device. A diafiltration process was designed to separate BSA from ovalbumin, with the Cibacron Blue recovered in a second stage operated with a buffer containing NaSCN to displace the dye from the protein. The results demonstrate the ability to use small charged affinity ligands with bio‐specific binding characteristics to enhance separations by high performance tangential flow filtration.

Viscoelasticity of concentrated emulsions stabilized by bovine serum albumin in the presence of a nonionic surfactant

Viscoelastic properties of highly concentrated toluene-in-water emulsions stabilized by mixtures of bovine serum albumin and a nonionic surfactant, polyoxyethylene(20)sorbitan monooleate, are studied by a dynamic method in a wide frequency range. It is shown that an increase in the relative content of the surfactant results in a suppression of the system elasticity and a decrease in the relaxation time. A direct correlation is found between the dynamic and steady rheological characteristics of the systems under examination. The latter fact confirms indirectly that the viscoelasticity of the emulsions is due to the presence of the high-molecular-mass component in interfacial layers.

Black foam films stabilized with the mixtures of bovine serum albumin and nonionic surfactant Tween 80

Stable black films are prepared for the first time from aqueous solutions of Tween 80-bovine serum albumin mixtures. Conditions for their formation and stabilization factors are studied in a Scheludko-Exerowa cell by microinterferometry. It is found that such films are formed when saturated surface layers consist of surfactant-protein 1: 1 complexes, Tween 80 molecules, or their mixtures. A possible mechanism for the thinning of liquid layers and the stabilization of black films is discussed.