Bovine Serum Albumin (bovine + serum_albumin)

Distribution by Scientific Domains
Distribution within Chemistry

Selected Abstracts

Bovine Serum Albumin and Lysozyme Adsorption on Calcium Phosphate Particles

Berit Mueller
Two model proteins that are oppositely charged at neutral pH , bovine serum albumin (BSA) and lysozyme, with acidic and alkaline isoelectric points, respectively , are used to investigate the protein adsorption behaviour of hydroxyapatite and beta-tricalcium phosphate (, -TCP) particles. Both calcium phosphate based particles are highly relevant for the fabrication of bioactive and resorbable bone implants. The investigations are carried out by combining zeta potential and Vis spectroscopy measurements. The changes of zeta potential and isoelectric point are determined as a function of added protein. Both proteins form a monolayer on , -TCP, while on hydroxyapatite only semi-monolayers were measured. For BSA, a side-on adsorption mode is suggested, whereas end-on adsorption appears to be most likely for lysozyme. The zeta potential curves as a function of adsorbed protein show that plateaus of the protein amounts adsorbed increase with charge saturation. In addition, the spatial charge distribution of both proteins is modelled to get a further understanding of the initial adsorption orientation of the biomolecules, supporting the findings from the experimental data. The reported findings can be transferred to the adsorption behaviour of a variety of proteins on calcium phosphate surfaces and are helpful for the fabrication of bone-analogous calcium phosphate/protein nanocomposites. [source]

Stabilization of PbS Nanocrystals by Bovine Serum Albumin in its Native and Denatured States

Mandeep Singh Bakshi
Abstract PbS nanocrystals (NCs) are synthesized in aqueous phase within a temperature range of 40,80,°C in the presence of native and denatured states of bovine serum albumen (BSA) as the capping/stabilizing agent. The NCs are characterized with the help of field-emission scanning electron microscopy, high-resolution transmission electron microscopy, X-ray diffraction, and energy-dispersive X-ray analysis. At 40,°C, large ball-shaped NCs (145,±,37,nm) with small surface protrusions are formed when 1,×,10,4,g mL,1 BSA is used. As the reaction temperature is increased towards 80,°C, the size of NCs decreases and they acquire somewhat cubic geometries (49.1,±,7.0,nm) due to a change in the capping behavior of BSA between its native and denatured states. The native and denatured states of BSA are simultaneously studied by fluorescence spectroscopy using tryptophan emission, and pH measurements with respect to time and temperature. Gel electrophoresis is used to determine the polarity of the BSA capped NCs. Only the small sized NCs conjugated with relatively larger amounts of BSA show a displacement towards the positively charged electrode in comparison to larger NCs with lower amounts of BSA capping. It was concluded that the denatured state of BSA is more effective in controlling the crystal growth of PbS than its native state especially in the low concentration range. [source]

Encapsulation and/or Release Behavior of Bovine Serum Albumin within and from Polylactide-Grafted Dextran Microspheres

Tatsuro Ouchi
Abstract Summary: Polylactide (PLA)-grafted dextran (Dex- graft -PLA) of various contents of sugar units was synthesized by anionic polymerization of L -lactide (L -LA) using the alkoxide of partially trimethylsilylated dextran (TMSDex) and subsequently removing the trimethylsilyl (TMS) groups. The copolymer showed different solubility from L -LA homopolymer with increasing the content of sugar units. We prepared bovine serum albumin (BSA)-loaded microspheres (MS)s according to a water-in-oil-in-water emulsion-solvent evaporation/extraction method using methylene chloride/DMSO as an organic cosolvent. MSs prepared from Dex- graft -PLA [MS(Dex- graft -PLA)s] exhibited higher loading efficiency of BSA than MSs prepared from PLLA [MS(PLLA)s]. The in vitro release rate of BSA from MS(Dex- graft -PLA) was faster than that from MS(PLLA). BSA released from MS(Dex- graft -PLA) maintained the secondary structure of native BSA to a great extent, compared with BSA released from MS(PLLA). Confocal fluorescence images of the differential interference micrographs over the fluorescence images of MS(PLLA) and MS(Dex- graft -PLA). [source]

A Novel Approach to Magnetic Nanoadsorbents with High Binding Capacity for Bovine Serum Albumin

Yabin Sun
Abstract Magnetic nanoadsorbents using Fe3O4 nanoparticles as cores and poly(methyl acrylic acid) (PMAA) as ionic exchange groups were prepared through our novel approach. Two steps were involved in this approach: the first was to functionalize the magnetic nanoparticles (MNPs) with methacrylate double bonds via the combination of ligand exchange and condensation of methacryloxypropyltrimethoxysilane(MPS); the second was to graft PMAA chains onto the surface of MNPs through radical polymerization. The success of the various surface functionalization steps was ascertained using FTIR and XPS. The as-synthesized PMAA-coated MNPs were effective in binding bovine serum albumin (BSA) with a high capacity of 1,300 mg,·,g,1. [source]

Molecular Recognition in Partially Folded States of a Transporter Protein: Temperature-dependent Specificity of Bovine Serum Albumin

Debapriya Banerjee
The specificity of molecular recognition of a transporter protein bovine serum albumin (BSA) in its different partially folded states has been studied. In order to avoid complications due to chemical denaturation, we have prepared thermally induced partially folded states of the protein. The partially folded states have been structurally characterized by circular dichroism and differential thermal analysis techniques. The change in the globular structure of the protein as a consequence of thermal unfolding has also been characterized by dynamic light scattering. Steady state, picosecond-resolved fluorescence and polarization gated spectroscopies on the ligands (DCM, LDS 750) in the protein reveal the dynamics of the binding sites and the specificity of ligand binding of BSA. Picosecond resolved Förster resonance energy transfer studies on the donor DCM and acceptor LDS 750 confirm that the specificity of ligand binding in the binding site is maintained up to 70°C. At 75°C, the protein loses its specificity of recognition at the aforesaid site. [source]

Photophysics in Motionally constrained Bioenvironment: Interactions of Norharmane with Bovine Serum Albumin,

Arabinda Mallick
ABSTRACT Steady-state photophysics of norharmane (NHM), a bioactive alkaloid, has been studied in the presence of a model transport protein, bovine serum albumin (BSA). The emission spectrum undergoes a remarkable change upon addition of BSA to the aqueous solution of NHM in buffer. Addition of BSA leads to a marked increase in the fluorescence anisotropy of the neutral species of NHM, although the fluorescence anisotropy for the cationic species is almost invariant to BSA addition, suggesting that the neutral species is located in a motionally restricted environment of BSA, whereas the cationic species remains in the bulk aqueous phase. The binding constant (K) and free energy change (,G) for the probe-protein binding have been calculated from the fluorescence data. Light has been thrown on the action of urea on protein-bound NHM. The denaturation study suggests that the protein, in its native form, binds with NHM. Polarity of the microenvironment around the probe has been determined from a comparison of the fluorescence properties of the two prototropic species of NHM in water-dioxane mixture with varying composition. [source]

Characterization of a Ruthenium(III)/NAMI-A Adduct with Bovine Serum Albumin that Exhibits a High Anti-Metastatic Activity,

Mimi Liu
Ru-ndum erneuert: Röntgenabsorptionsspektroskopische Studien belegen, dass der RuIII -Wirkstoff NAMI-A alle seine Cl, - und S-Donor-Liganden gegen N-Donoren und Carboxylatogruppen von Rinderserumalbumin (BSA) austauschen kann (siehe Schema). Das RuIII -BSA-Addukt verstärkt Zell-Substrat-Wechselwirkungen ebenso stark wie NAMI-A (das sich im Zellkulturmedium binnen Minuten in RuIII -BSA umwandelt). [source]

Synthesis of Gold Microplates Using Bovine Serum Albumin as a Reductant and a Stabilizer

Leslie Au
Abstract Gold microplates were synthesized in aqueous solutions by reducing HAuCl4 with the hydroxyl groups in both serine and threonine of bovine serum albumin (BSA), which is a globular protein in its native state. In this article, we systematically investigated the effects of temperature, pH value, the concentration of BSA, and ionic species on the reduction kinetics and thus the size and morphology of the final product. The optimal experimental conditions for producing uniform Au microplates include the following: an elevated temperature in the range of 55,65,°C, an acidic solution with pH,3, and the presence of NaCl (0.14,M). We found that if any one of these parameters was deviated from the optimal condition, Au microplates would not be formed in high yields. We also found that the surfaces of the as-synthesized Au microplates were covered by a dense array of BSA bumps. [source]

Conformational Stability of Bovine Serum Albumin in Aqueous Amides: A Further Insight into the Mechanism of Urea Acting on the Protein

Lin Ma
Abstract The binding distances of fluorescein to bovine serum albumin (BSA) in formamide-water and N,N -dimethyl- formamide-water mixtures were determined by fluorescence quenching method and compared with the values in urea-water mixtures in our previous work. The results, together with the analysis of fluorescence spectra, were utilized to probe the conformational stability of protein in aqueous amides, providing a further insight into the mechanism of urea acting on protein. The spectral properties of BSA showed significant difference in the aqueous solutions of the three kinds of amide and indicated that both NH2 group and C=O group could form hydrogen bond with the protein, serving as donor and acceptor, respectively. However, the results revealed that the multiple hydrogen bonds of NH2 group with back bond and hydrophilic side chains of the protein played a key role in the nonspecific urea-mediated network of intramolecular interaction due to its higher hydrogen bonding capability compared to C=O group. [source]

Interaction of Co(II) with Bovine Serum Albumin under UV C Irradiation

He-Dong Zhang
Abstract The interaction of Co(II) with BSA under UV C (253.7 nm) irradiation under physiological conditions has been studied by UV-vis spectrum, ultraviolet second-derivative spectroscopy and fluorescence spectrum. The quenching rate constant kq and the association constant Ka were calculated according to Stern-Volmer equation based on the quenching of the fluorescence of BSA by Co(II). [source]

Study on the Interaction of Ketoconazole with Human and Bovine Serum Albumins by Fluorescence Spectroscopy

Qing-Lian GUO
Abstract The binding of ketoconazole to human serum albumin and bovine serum albumin was studied by using fluorescence and ultraviolet spectroscopy. The measurements were performed in 0.1 mol·L,1 phosphate buffer solution at pH=7.40±0.1. Decreasing of quenching constant was observed in association with temperature increase. Our findings show that the quenching mechanism of fluorescence of serum albumins by ketoconazole was static quenching because of compound formation. The thermodynamic parameters ,G, ,H, and ,S at different temperatures were calculated, showing that the electrostatic interactions and hydrophobic interaction are the main forces for the binding of ketoconazole to serum albumins. The distance r between the donor (Trp-214) and acceptor (ketoconazole) was obtained according to fluorescence resonance energy transfer theory. [source]

Photopolymerizable Hydrogels Made from Polymer-Conjugated Albumin for Affinity-Based Drug Delivery,

Liat Oss-Ronen
As a drug delivery vehicle, biodegradable albumin hydrogels can combine the high binding capacity of albumin with the structural stability of a polymeric hydrogel network to enable controlled release of small molecules based on both binding affinity and physical interactions. In the present study, we report on the development of a hybrid hydrogel composed of albumin conjugated to poly(ethylene glycol) (PEG) for drug delivery applications where controlled release is accomplished using the natural affinity of the drugs to the serum albumin. Bovine serum albumin was conjugated to PEG-diacrylate having a molecular weight of 1.5, 4, or 10,kDa to form a PEGylated albumin macromolecule (mono-PEGylated or multi-PEGylated). Biodegradable hydrogels were formed from the PEGylated albumin using photopolymerization. Two model drugs, Warfarin and Naproxen, were used for equilibrium dialysis and release experiments from the hydrogels, both having relatively low molecular weights and a known high affinity for albumin. Equilibrium dialysis experiments showed that multi-PEGylation of albumin significantly decreased the drug affinity to the protein compared to non-PEGylated controls, irrespective of the PEG molecular weight. However, the results from drug release experiments showed that mono-PEGylation of albumin did not change its natural affinity to the drug. Comparing the release profiles with a Fickian diffusion model provided strong evidence that hydrogels containing mono-PEGylated albumin exhibited sub-diffusive drug release properties based on the affinity of the drug to the tethered protein. [source]

Preparation and in vitro evaluation of new pH-sensitive hydrogel beads for oral delivery of protein drugs

I. M. El-Sherbiny
Abstract New biodegradable pH-responsive hydrogel beads based on chemically modified chitosan and sodium alginate were prepared and characterized for the controlled release study of protein drugs in the small intestine. The ionotropic gelation reaction was carried out under mild aqueous conditions, which should be appropriate for the retention of the biological activity of an uploaded protein drug. The equilibrium swelling studies were carried out for the hydrogel beads at 37°C in simulated gastric (SGF) and simulated intestinal (SIF) fluids. Bovine serum albumin (BSA), a model for protein drugs was entrapped in the hydrogels and the in vitro drug release profiles were established at 37°C in SGF and SIF. The preliminary investigation of the hydrogel beads prepared in this study showed high entrapment efficiency (up to 97%) and promising release profiles of BSA. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2010 [source]

Application of ultrasonic shear rheometer to characterize rheological properties of high protein concentration solutions at microliter volume

Atul Saluja
Abstract The purpose of this work was to conduct preliminary rheological analysis on high protein concentration solutions by using the technique of ultrasonic shear rheometry at megahertz frequencies. The work was aimed at establishing the viability of the technique for analyzing protein solution rheology as well as obtaining an initial understanding of the effect of solution conditions on solution rheology of a model protein. Bovine serum albumin (BSA) was used for this study, and rheological analysis was conducted at 20 ,L sample volume between pH 2.0 and 9.0 at different ionic strengths at 25°C using 5 and 10 MHz quartz crystals. Significant differences in storage modulus among solutions at pH 5.0, 7.0, and 9.0 could only be detected at 10 MHz, and the errors associated with measurements were smaller as compared to those at 5 MHz for all the solutions studied. Solutions at pH 2.0 and 3.0 showed a time-dependent change in solution rheology. For solutions at pH 5.0, 7.0, and 9.0, which did not show time dependence in solution rheology, loss modulus data at lower concentrations correlated well with the dilute solution data in the literature. At higher concentrations, pH 5.0 solutions exhibited a higher loss modulus than pH 7.0 and pH 9.0 solutions. Storage modulus decreased with increasing ionic strength, unlike loss modulus, which did not show any change, except at pI of protein when no effect was observed. The results show the potential of high frequency rheometry for analyzing subtle differences in rheology of pharmaceutically relevant protein solutions at microliter volume. © 2005 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 94:1161,1168, 2005 [source]

Albumin enhanced morphometric image analysis in CLL,

CYTOMETRY, Issue 1 2004
Matthew A. Lunning
Abstract BACKGROUND The heterogeneity of lymphocytes from patients with chronic lymphocytic leukemia (CLL) and blood film artifacts make morphologic subclassification of this disease difficult. METHODS We reviewed paired blood films prepared from ethylene-diamine-tetraacetic acid (ETDA) samples with and without bovine serum albumin (BSA) from 82 CLL patients. Group 1 adhered to NCCLS specifications for the preparations of EDTA blood films. Group 2 consisted of blood films containing EDTA and a 1:12 dilution of 22% BSA. Eight patients were selected for digital photomicroscopy and statistical analysis. Approximately 100 lymphocytes from each slide were digitally captured. RESULTS The mean cell area ± standard error was 127.8 ,m2 ± 1.42 for (n = 793) for group 1 versus 100.7 ,m2 ± 1.39 (n = 831) for group 2. The nuclear area was 88.9 ,m2 ± 0.85 for group 1 versus 76.4 ,m2 ± 0.83 for group 2. For the nuclear transmittance, the values were 97.6 ± 0.85 for group 1 and 104.1 ± 0.83 for group 2. The nuclear:cytoplasmic ratios were 0.71 ± 0.003 for group 1 and 0.78 ± 0.003 for group 2. All differences were statistically significant (P < 0.001). CONCLUSIONS BSA addition results in the reduction of atypical lymphocytes and a decrease in smudge cells. BSA also decreases the lymphocyte area and nuclear area, whereas nuclear transmittance and nuclear:cytoplasmic ratio are increased. A standardized method of slide preparation would allow accurate interlaboratory comparison. The use of BSA may permit better implementation of the blood film-based subclassification of CLL and lead to a better correlation of morphology with cytogenetics and immunophenotyping. Published 2003 Wiley-Liss, Inc. [source]

An actin-stabilizing peptide conjugate deduced from the major outer sheath protein of the bacterium Treponema denticola

CYTOSKELETON, Issue 9 2007
Mohsen Amin
Abstract A synthetic peptide conjugated to bovine serum albumin, P34BSA, based on a 10-mer in the deduced amino acid sequence of the major outer sheath protein of Treponema denticola, was found to stabilize actin filaments of fibroblasts. Pretreatment of cells with P34BSA inhibited the actin disruption induced by cytochalasin D and latrunculin B. P34BSA was taken up by the cells and localized among actin filaments. P34BSA bound actin from fibroblast lysates, and cell exposure to P34BSA led to the activation of RhoA, a key regulator of actin filament assembly in fibroblasts. Exposure of fibroblasts to P34BSA retarded their migration on a collagen substratum. P34BSA also inhibited chemotaxis of murine neutrophils. Our findings with a novel peptide conjugate imply that bacterial proteins known to perturb the cytoskeleton represent a rich source of molecular models upon which to design synthetic reagents for modulating actin-dependent cellular functions. Cell Motil. Cytoskeleton 2007. © 2007 Wiley-Liss, Inc. [source]

Ca2+ -dependent in vitro contractility of a precipitate isolated from an extract of the heliozoon Actinophrys sol

CYTOSKELETON, Issue 2 2006
Mikihiko Arikawa
Abstract Contraction of axopodia in actinophrid heliozoons (protozoa) is induced by a unique contractile structure, the "contractile tubules structure (CTS)". We have previously shown that a cell homogenate of the heliozoon Actinophrys sol yields a precipitate on addition of Ca2+ that is mainly composed of filamentous structures morphologically identical to the CTS. In this study, to further characterize the nature of the CTS in vitro, biochemical and physiological properties of the precipitate were examined. SDS-PAGE analysis showed that the Ca2+ -induced precipitate was composed of many proteins, and that no proteins in the precipitate showed any detectable changes in electrophoretic mobility on addition of Ca2+. Addition of extraneous proteins such as bovine serum albumin to the cell homogenate resulted in cosedimentation of the proteins with the Ca2+ -induced precipitate, suggesting that the CTS has a high affinity for other proteins that are not related to precipitate formation. Appearance and disappearance of the precipitate were repeatedly induced by alternating addition of Ca2+ and EGTA, and its protein composition remained unchanged even after repeated cycles. When adhered to a glass surface, the precipitate showed Ca2+ -dependent contractility with a threshold of 10,100 nM, and this contractility was not inhibited by colchicine or cytochalasin B. The precipitate repeatedly contracted and relaxed with successive addition and removal of Ca2+, indicating that the contraction was controlled by Ca2+ alone with no need for any other energy supply. From our characterization of the precipitate, we concluded that its Ca2+ -dependent formation and contraction are associated with the unique contractile organelle, the "contractile tubules structure". Cell Motil. Cytoskeleton 2006. © 2005 Wiley-Liss, Inc. [source]

Lactoferrin and anti-lactoferrin antibodies: Effects of ironloading of lactoferrin on albumin extravasation in different tissues in rats

Lactoferrin is a cationic iron-binding protein, which is released from activated neutrophils in concert with reactive oxygen species. In vitro, lactoferrin has both anti- and proinflammatory effects; many of them dependent on iron-binding. In vivo, only iron-free lactoferrin reduced inflammatory hyperpermeability in the lung. We therefore examined whether 1 mg iron-free (Apo-Lf) or iron-saturated lactoferrin (Holo-Lf) alone or followed by anti-lactoferrin antibodies (aLf) affected permeability evaluated by extravasation of radiolabelled bovine serum albumin (CBSA) in different tissues of anaesthetized rats. Fifteen minutes after i.v. injection of Lf, aLf or saline was given and circulatory arrest was induced 20 min thereafter. Measurements were performed in control, after Apo-Lf, Holo-Lf, Apo-Lf + aLf, Holo-Lf + aLf and aLf alone (n=6,8 in each group). No intergroup differences were found for plasma volume and haematocrit at the start and end of the 37 min extravasation period or for total tissue water in any of the six different tissues studied, excluding larger transcapillary fluid shifts. However, increases in CBSA were seen without differences in tissue intravascular volume. Iron-free lactoferrin and aLf alone did not change CBSA significantly. Iron-saturated lactoferrin significantly increased CBSA in skin (neck), trachea and left ventricle of the heart to 249 ± 9, 284 ± 16 and 160 ± 7% of control, respectively. When followed by aLf, both Apo- and Holo-Lf increased CBSA significantly in four and five of the tissues studied, respectively. However, no significant effect was seen for Holo-Lf + aLf compared with Holo-Lf alone. In conclusion, iron-saturated, but not iron-free lactoferrin increased CBSA, whereas antilactoferrin increased CBSA compared with lactoferrin alone only when following iron-free lactoferrin. [source]

Voltammetric Studies of the Interactions Between Ferrocene-Labeled Glutathione and Proteins in Solution or Immobilized onto Surface

Yong Peng
Abstract Glutathione (GSH) tagged with a ferrocene (Fc) label at its C-terminal was synthesized via coupling ferrocenyl amine to glutathione using o -(benzotriazol-1-yl)- N,N,N,,N, -tetramethyluronium (HBTU)/1-hydroxybenzotrizole (HOBt). The presence of Fc yielded well defined voltammetric signals, rendering the Fc-tagged GSH (GSH-Fc) suitable for electrochemical studies of GSH binding to other biological species. The interaction of GSH-Fc with bovine serum albumin (BSA) was investigated, and a binding ratio of 1.41±0.06 (GSH-Fc/BSA) and an affinity constant Ka of 6.53±2.01×106,M,1 were determined. These results compare well with those measured by fluorescence using untagged GSH, suggesting that the attachment of Fc to GSH does not significantly perturb the GSH structure and binding behavior. By contrasting the binding behavior to several compounds that are known to conjugate to different domains of BSA, the voltammetric study confirmed that GSH-Fc binds at subdomain IIA of BSA with high affinity. The versatility of GSH-Fc for studying GSH binding to surface-confined proteins was also demonstrated with the GSH binding to electroinactive Zn-metallothionein (Zn7 -MT) through hydrogen binding at the region between the Zn7 -MT , and , domains. [source]

Electrochemistry of Mitochondria: A New Way to Understand Their Structure and Function

Jing Zhao
Abstract In this article, electrochemistry of mitochondria is achieved. Cyclic voltammograms of freshly prepared mitochondria were obtained by immobilizing mitochondria together with glutaraldehyde and bovine serum albumin on the surface of a pyrolytic graphite electrode. Two pairs of redox peaks could be observed which were ascribed to the electron transfer reactions of cytochrome c and FAD/FADH2. Study of submitochondrial particles was also conducted, which could confirm the results of the study of the entire mitochondria. The redox wave of NADH could be obtained due to the destruction of the membrane of mitochondria. We have also checked the function of succinate in mitochondria by employing the electrochemical method. This work is not only the first to be able to obtain the direct electrochemistry of mitochondria, but is also beneficial to the further understanding of the structure and function of mitochondria in vitro. [source]

Glucose Biosensor Mediated by 1,2-Diferrocenylethane in a Sono-Gel Composite Electrode

Barbara Ballarin
Abstract An amperometric glucose biosensor was constructed based on a renewable carbon composite sono-gel matrix incorporating 1,2-diferrocenylethane as electron transfer mediator between the electrode and the active site of glucose oxidase. The enzyme was immobilized on the electrode surface by cross-linking with glutaraldehyde and bovine serum albumin. The process parameters for the fabrication of the biosensor and the influence of various experimental conditions (i.e., pH, temperature, operating potential) were investigated. Cyclic voltammetry and amperometric measurements were used to study the response of the glucose sensor, which displayed fast response time and good reproducibility. The analytical performances and the apparent Michaelis-Menten constant of the biosensor were evaluated. [source]

Rapid separation of protein isoforms by capillary zone electrophoresis with new dynamic coatings

William W. P. Chang
Abstract Many cellular functions are regulated through protein isoforms. Changes in the expression level or regulatory dysfunctions of isoforms often lead to developmental or pathological disorders. Isoforms are traditionally analyzed using techniques such as gel- or capillary-based isoelectric focusing. However, with proper electroosmotic flow (EOF) control, isoforms with small pI differences can also be analyzed using capillary zone electrophoresis (CZE). Here we demonstrate the ability to quickly resolve isoforms of three model proteins (bovine serum albumin, transferrin, ,1 -antitrypsin) in capillaries coated with novel dynamic coatings. The coatings allow reproducible EOF modulation in the cathodal direction to a level of 10 -9 m2V -1s -1. They also appear to inhibit protein adsorption to the capillary wall, making the isoform separations highly reproducible both in peak areas and apparent mobility. Isoforms of transferrin and ,1 -antitrypsin have been implicated in several human diseases. By coupling the CZE isoform separation with standard affinity capture assays, it may be possible to develop a cost-effective analytical platform for clinical diagnostics. [source]

A microfabricated capillary electrophoresis chip with multiple buried optical fibers and microfocusing lens for multiwavelength detection

Suz-Kai Hsiung
Abstract We present a new microfluidic device utilizing multiwavelength detection for high-throughput capillary electrophoresis (CE). In general, different fluorescent dyes are only excited by light sources with appropriate wavelengths. When excited by an appropriate light source, a fluorescent dye emits specific fluorescence signals of a longer wavelength. This study designs and fabricates plastic micro-CE chips capable of performing multiple-wavelength fluorescence detection by means of multimode optic fiber pairs embedded downstream of the separation channel. For detection purposes, the fluorescence signals are enhanced by positioning microfocusing lens structures at the outlets of the excitation fibers and the inlets of the detection fibers, respectively. The proposed device is capable of detecting multiple samples labeled with different kinds of fluorescent dyes in the same channel in a single run. The experimental results demonstrate that various proteins, including bovine serum albumin and ,-casein, can be successfully injected and detected by coupling two light sources of different wavelengths to the two excitation optic fibers. Furthermore, the proposed device also provides the ability to measure the speed of the samples traveling in the microchannel. The developed multiwavelength micro-CE chip could have significant potential for the analysis of DNA and protein samples. [source]

Multilayer poly(vinyl alcohol)-adsorbed coating on poly(dimethylsiloxane) microfluidic chips for biopolymer separation

Dapeng Wu
Abstract A poly(dimethylsiloxane) (PDMS) microfluidic chip surface was modified by multilayer-adsorbed and heat-immobilized poly(vinyl alcohol) (PVA) after oxygen plasma treatment. The reflection absorption infrared spectrum (RAIRS) showed that 88% hydrolyzed PVA adsorbed more strongly than 100% hydrolyzed one on the oxygen plasma-pretreated PDMS surface, and they all had little adsorption on original PDMS surface. Repeating the coating procedure three times was found to produce the most robust and effective coating. PVA coating converted the original PDMS surface from a hydrophobic one into a hydrophilic surface, and suppressed electroosmotic flow (EOF) in the range of pH 3,11. More than 1 000,000 plates/m and baseline resolution were obtained for separation of fluorescently labeled basic proteins (lysozyme, ribonuclease B). Fluorescently labeled acidic proteins (bovine serum albumin, ,-lactoglobulin) and fragments of dsDNA ,X174 RF/HaeIII were also separated satisfactorily in the three-layer 88% PVA-coated PDMS microchip. Good separation of basic proteins was obtained for about 70 consecutive runs. [source]

A systematic investigation into the recovery of radioactively labeled proteins from sodium dodecyl sulfate-polyacrylamide gels

Shaobo Zhou
Abstract We report the results of a systematic investigation designed to optimize a method for quantifying radioactivity in proteins in sodium dodecyl sulfate-polyacrylamide gels. The method involves dissolving appropriately sized pieces of gel in hydrogen peroxide and heating to 70°C overnight followed by liquid scintillation counting. H2O2 had no effect on the count rates of [14C]bovine serum albumin (BSA) when counted in a conventional liquid scintillation system, and the count rates remained stable for several days. Temperatures below 70°C resulted in incomplete extraction of radioactivity from gels containing [14C]BSA, but there was also a significant reduction in count rates in samples incubated at 80°C. At 70°C recovery was not affected by the amount of sample loaded onto the gel or by the staining procedure (Coomassie Brilliant Blue or SYPRO Ruby). Recoveries were in the range of 89,94%, and the coefficient of variation for five replicate samples was 5,10%. This method offers a reliable way of measuring the amount of radioactivity in proteins that have been separated by electrophoresis. It may be useful, for example, in quantitative metabolic labeling experiments when it is necessary to know precisely how much tracer has been incorporated into a particular protein. [source]

High-efficiency peptide analysis on monolithic multimode capillary columns: Pressure-assisted capillary electrochromatography/capillary electrophoresis coupled to UV and electrospray ionization-mass spectrometry

Alexander R. Ivanov
Abstract High-efficiency peptide analysis using multimode pressure-assisted capillary electrochromatography/capillary electrophoresis (pCEC/pCE) monolithic polymeric columns and the separation of model peptide mixtures and protein digests by isocratic and gradient elution under an applied electric field with UV and electrospray ionization-mass spectrometry (ESI-MS) detection is demonstrated. Capillary multipurpose columns were prepared in silanized fused-silica capillaries of 50, 75, and 100 ,m inner diameters by thermally induced in situ copolymerization of methacrylic monomers in the presence of n -propanol and formamide as porogens and azobisisobutyronitrile as initiator. N -Ethylbutylamine was used to modify the chromatographic surface of the monolith from neutral to cationic. Monolithic columns were termed as multipurpose or multimode columns because they showed mixed modes of separation mechanisms under different conditions. Anion-exchange separation ability in the liquid chromatography (LC) mode can be determined by the cationic chromatographic surface of the monolith. At acidic pH and high voltage across the column, the monolithic stationary phase provided conditions for predominantly capillary electrophoretic migration of peptides. At basic pH and electric field across the column, enhanced chromatographic retention of peptides on monolithic capillary column made CEC mechanisms of migration responsible for separation. The role of pressure, ionic strength, pH, and organic content of the mobile phase on chromatographic performance was investigated. High efficiencies (exceeding 300,000 plates/m) of the monolithic columns for peptide separations are shown using volatile and nonvolatile, acidic and basic buffers. Good reproducibility and robustness of isocratic and gradient elution pressure-assisted CEC/CE separations were achieved for both UV and ESI-MS detection. Manipulation of the electric field and gradient conditions allowed high-throughput analysis of complex peptide mixtures. A simple design of sheathless electrospray emitter provided effective and robust low dead volume interfacing of monolithic multimode columns with ESI-MS. Gradient elution pressure-assisted mixed-mode separation CE/CEC-ESI-MS mass fingerprinting and data-dependent pCE/pCEC-ESI-MS/MS analysis of a bovine serum albumin (BSA) tryptic digest in less than 5 min yielding high sequence coverage (73%) demonstrated the potential of the method. [source]

Surface grafting of glycidyl methacrylate on silica gel and polyethylene beads

Seong-Ho Choi
Abstract Surface grafting of glycidyl methacrylate (GMA) on silica gel and a polyethylene bead was performed by radical polymerization and radiation-induced polymerization, respectively, in order to improve softness. Subsequently, diethylene triamine (DETA), triethylene tetraamine (TETA), and iminodiacetic acid (IDA) were introduced to the grafted GMA for use as affinity columns. The efficiency of the affinity column was investigated by use of bovine serum albumin (BSA) and hemoglobin (Hb) as model proteins. The affinity degree of BSA was higher than Hb for the DETA and TETA column, whereas the affinity degree of Hb was higher than BSA for the IDA column supported by silica gel. The affinity degree of BSA was higher than Hb for the DETA and TTA column supported by polyethylene (PE) beads. [source]

Spectroscopic investigation of the function of aqueous 2-hydroxyethylmethacrylate/glutaraldehyde solution as a dentin desensitizer

Chuangye Qin
Fourier-transform (FT)-Raman and -infrared (IR) spectroscopy were employed to investigate the function of the aqueous 2-hydroxyethylmethacrylate/glutaraldehyde solution (Gluma) as a desensitizer. 2-Hydroxyethylmethacrylate (HEMA), glutaraldehyde (GA), and the mixture of HEMA/GA (i.e. Gluma) were used to interact with dentin, collagen, hydroxyapatite (HAP), and bovine serum albumin (BSA) individually. All the interactions were monitored by an FT-Raman spectrometer. FT-IR spectroscopy was also used in this study. The results show that HEMA could be absorbed by dentin and collagen; GA could cross-link collagen and BSA; and when BSA was added to Gluma, polymerization of HEMA occurred. The results suggest that Gluma acts as a desensitizer whereby, first, GA reacts with part of the serum albumin in dentinal fluid, which induces a precipitation of serum albumin, then, second, a reaction of GA with serum albumin induces polymerization of HEMA. The function of Gluma as a desensitizer to block dentinal tubules occurs via these two reactions. [source]

Bovine Serum Albumin and Lysozyme Adsorption on Calcium Phosphate Particles

Berit Mueller
Two model proteins that are oppositely charged at neutral pH , bovine serum albumin (BSA) and lysozyme, with acidic and alkaline isoelectric points, respectively , are used to investigate the protein adsorption behaviour of hydroxyapatite and beta-tricalcium phosphate (, -TCP) particles. Both calcium phosphate based particles are highly relevant for the fabrication of bioactive and resorbable bone implants. The investigations are carried out by combining zeta potential and Vis spectroscopy measurements. The changes of zeta potential and isoelectric point are determined as a function of added protein. Both proteins form a monolayer on , -TCP, while on hydroxyapatite only semi-monolayers were measured. For BSA, a side-on adsorption mode is suggested, whereas end-on adsorption appears to be most likely for lysozyme. The zeta potential curves as a function of adsorbed protein show that plateaus of the protein amounts adsorbed increase with charge saturation. In addition, the spatial charge distribution of both proteins is modelled to get a further understanding of the initial adsorption orientation of the biomolecules, supporting the findings from the experimental data. The reported findings can be transferred to the adsorption behaviour of a variety of proteins on calcium phosphate surfaces and are helpful for the fabrication of bone-analogous calcium phosphate/protein nanocomposites. [source]

Diastereoselective Synthesis of -Hydroxy Sulfoxides: Enzymatic and Biomimetic Approaches

Stefano Colonna
Abstract Stereoselectivities of up to 98,% have been found in the enzymatic synthesis of ,-hydroxy sulfoxides catalyzed by cyclohexanone monooxygenase (CHMO). The diastereoselectivity of the "one-pot" preparation of the title compounds in the presence of bovine serum albumin has also been investigated. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007) [source]