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PubMed2006

Spectrophotometric determination of acidity constants of alizarine red S in water, water-Brij-35 and water-SDS micellar media solutions.

The acidity constants of Alizarine Red S in water, water-Brij-35 and water-SDS micellar media solutions at 25 degrees C and an ionic strength of 0.1 M have been determined spectrophotometrically. To evaluate the pH-absorbance data, a resolution method based on the combination of soft- and hard-modeling is applied. The acidity constants of all related equilibria are estimated using the whole spectral fitting of the collected data to an established factor analysis model. DATAN program applied for determination of acidity constants. Results show that the pKa values of Alizarine Red S are influenced as the percentages of a neutral and an anionic surfactant such as Brij-35 and SDS, respectively, added to the solution of this reagent. Effect of surfactant on acidity constants and pure spectrum of each component are also discussed.

Anthraquinones↗

PubMed2002

Structural changes are induced in human neutrophil cytochrome b by NADPH oxidase activators, LDS, SDS, and arachidonate: intermolecular resonance energy transfer between trisulfopyrenyl-wheat germ agglutinin and cytochrome b(558).

Anionic amphiphiles such as sodium- and lithium dodecyl sulfate (SDS, LDS), or arachidonate (AA) initiate NADPH oxidase and proton channel activation in cell-free systems and intact neutrophils. To investigate whether these amphiphiles exert allosteric effects on cytochrome b, trisulfopyrenyl-labeled wheat germ agglutinin (Cascade Blue-wheat germ agglutinin, CCB-WGA) was used as an extrinsic fluorescence donor for resonance energy transfer (RET) to the intrinsic heme acceptors of detergent-solubilized cytochrome b. In solution, cytochrome b complexed with the CCB-WGA causing a rapid, saturable, carbohydrate-dependent quenching of up to approximately 55% of the steady-state fluorescence. Subsequent additions of SDS, LDS, or AA to typical cell-free oxidase assay concentrations completely relaxed the fluorescence quenching. The relaxation effects were specific, and not caused by dissociation of the CCB-WGA-cytochrome b complex or alterations in the spectral properties of the chromophores. In contrast, addition of the oxidase antagonist, arachidonate methyl ester, caused an opposite effect and was able to partially reverse the activator-induced relaxation. We conclude that the activators induce a cytochrome b conformation wherein the proximity or orientation between the hemes and the extrinsic CCB fluorescence donors has undergone a significant change. These events may be linked to NADPH oxidase assembly and activation or proton channel induction.

Arachidonic Acid↗

PubMed1983

A sensitive SDS-PAGE method separating myosin heavy chain isoforms of rat skeletal muscles reveals the heterogeneous nature of the embryonic myosin.

Myosin isoforms are used as markers of heterogeneity and plasticity of skeletal muscle fibers and motor units. Tedious and time-consuming methods, needing microgram or milligram amounts of myosin are widely used to characterize the heavy subunits. We here describe a sensitive method that separates in nanogram or microgram amounts the heavy chains of immature, fast and slow adult rat muscles in complex mixtures of myosins. Though the method is assembled from published procedures (SDS-PAGE, peptide mapping in the presence of SDS, silver stain) for the logical extensions introduced the end-product is a powerful tool to separate and characterize these high molecular weight biopolymers until now inseparable from complex mixtures. The method reveals the heterogeneous nature of the embryonic myosin heavy chains.

Animals↗

PubMed2003

Spreading of aqueous SDS solutions over nitrocellulose membranes.

Experimental investigations were carried out on the spreading of small drops of aqueous SDS solutions over dry thin porous substrates (nitrocellulose membranes) in the case of partial wetting. The time evolution was monitored of the radii of both the drop base and the wetted area inside the porous substrate. The total duration of the spreading process was subdivided into three stages: the first stage: the drop base expands until the maximum value of the drop base is reached, the contact angle rapidly decreases during this stage; the second stage: the radius of the drop base remains constant and the contact angle decreases linearly with time; the third stage: the drop base shrinks and the contact angle remains constant. The wetted area inside the porous substrate expends during the whole spreading process. Appropriate scales were used with a plot of the dimensionless radii of the drop base, of the wetted area inside the porous substrate and the dynamic contact angle on the dimensionless time. Our experimental data show: the overall time of the spreading of drops of SDS solution over dry thin porous substrates decreases with the increase of surfactant concentration; the difference between advancing and hydrodynamic receding contact angles decreases with the surfactant concentration increase; the constancy of the contact angle during the third stage of spreading has nothing to do with the hysteresis of contact angle, but determined by the hydrodynamic reasons. It is shown using independent spreading experiments of the same drops on nonporous nitrocellulose substrate that the static receding contact angle is equal to zero, which supports our conclusion on the hydrodynamic nature of the hydrodynamic receding contact angle on porous substrates.

Collodion↗

PubMed2003

Testing the Gouy-Chapman theory by means of surface tension measurements for SDS-NaCl-H2O mixtures.

Surface tension isotherms were measured for sodium dodecyl sulfate (SDS) at different concentrations of added salt (NaCl). The free energy of the surfactant monolayer was assessed by invoking the Gouy-Chapman theory for the charged head groups, the hydrophobic (Tanford) free energy of transfer of the hydrocarbon chain, and the hydrocarbon chain configurational free energy according to Gruen's calculations and finally macroscopic contact terms. In particular, the effect of an increased salt concentration in bulk was examined. Theoretical predictions compare well with the experimental findings, and good agreement was found with respect to both the variation of free energy of the monolayer and the surface pressure behavior. Thus, at least for a liquid-expanded monolayer of SDS, the Gouy-Chapman model yields a satisfactory account of the electrostatic contribution to the thermodynamic properties at different salt concentrations of NaCl.

Journal Article↗

PubMed1997

Multiple immunoblots after non-electrophoretic bidirectional transfer of a single SDS-PAGE gel with multiple antigens.

Western blotting is a very sensitive and powerful fundamental technique in immunology that has been used to detect and characterize proteins of low abundance. This technique employs the transfer of proteins separated on SDS-PAGE to nitrocellulose sheets for further detection using antibodies. Here we report the non-electrophoretic transfer of the 60-kDa Ro (or SSA) autoantigen, 240 and 220 kDa spectrin antigens and prestained molecular weight standards from SDS-PAGE gels to nitrocellulose to obtain multiple immunoblots. In fact, we have used this procedure to obtain 12 immunoblots from a single gel with multiple sera.

Antigens↗

PubMed1988

Evaluation of a computer-assisted method of analysing SDS-PAGE protein profiles in tracing a hospital outbreak of Serratia marcescens.

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles of bacterial proteins have been successfully used for taxonomical purposes. More recently this technique has been applied to epidemiological investigations in respect of various micro-organisms including Neisseria meningitidis, Staphylococcus aureus and Clostridium difficile. The main limitations of the methods so far described are lack of standardisation in extraction and separation as well as in the analysis of results. Although reproducibility in the same laboratory has been shown to be satisfactory, comparison of results among laboratories is still difficult. Moreover, assessment of differences and/or similarities among chromatograms or autoradiographs showing many bands depends upon qualitative descriptions. Interpretation of densitometric scannings is laborious and time-consuming. In this paper we present our experience of a completely standardised, fully computer-controlled procedure for SDS-PAGE (AMBIS System) in analysing 35S-methionine-labelled total proteins. The methodology proved very useful in monitoring a hospital outbreak of Serratia marcescens. It allowed us to make quantitative comparison in a shorter time as well as to handle easily a great amount of data and usefully integrate it with those obtained with other systems such as serotyping. Furthermore, when the two systems are used together, more precise information can be gained. In this epidemic, serotyping indicated the presence of two groups which would have been missed by PAGE analysis alone. Electrophoretotyping, however, focused on similarities of cellular proteins among the epidemic strains. This allowed us to distinguish them from epidemiologically unrelated strains of the same serogroup.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Infections↗

PubMedDate not supplied

A comparison between SDS-PAGE and size exclusion chromatography as analytical methods for determining product composition in protein conjugation reactions.

Horseradish peroxidase (HRP) was conjugated with bovine serum albumin (BSA) or human alpha(1)-proteinase inhibitor (alpha(1)-PI). The enzyme was maleimidylated using N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and then allowed to react with thiolated BSA or reduced alpha(1)-PI. The conjugation products were analysed both by SDS-PAGE and size exclusion chromatography (SEC) on Sephadex G200. The two methods of evaluating conjugative processes were compared with respect to information provided in relation to the behaviour of the products in solution. The results showed that neither SDS-PAGE nor SEC alone provides sufficient information about conjugate structure. The basic conjugate units observed in electrophoresis tend to form dimeric or higher-order aggregates under gel chromatographic conditions.

Animals↗

PubMed2002

Detection of aminotransferase activity of Propionibacterium freudenreichii after SDS-PAGE.

Aminotransferases (ATs) had previously been detected after native electrophoresis. We show now that aminotransferase(s) of Propionibacterium freudenreichii can be detected after sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Moreover, it retained a high activity (84%) in the presence of 0.23% SDS, contrary to what was observed for aminotransferase(s) of Bifidobacterium bifidum (54%) and of six other cheese-related species (0-20%).

Bifidobacterium↗

PubMed2003

Identification of lactic acid bacteria from spontaneous fermentation of 'Almagro' eggplants by SDS-PAGE whole cell protein fingerprinting.

In order to complete the previously performed phenotypic identification [Sánchez et al., 2000. Int. J. Food Microbiol. 59 (2000) 9], whole cell protein patterns obtained using Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) of 149 lactic acid bacteria (LAB) strains, isolated from 'Almagro' eggplants spontaneous fermentation, were analysed. Phenotypic identification of those strains had revealed the presence of the species Lactobacillus (Lb) plantarum, Lb. pentosus, Lb. brevis biotype 2, Lb. brevis biotype 3, Lb. fermentum, Lactobacillus spp. and Aerococcus viridans. The SDS-PAGE technique generated complex and stable patterns that were easy to interpret and compare with those of the 17 reference strains used in this study. After numerical analysis of the resulting electrophoretic protein patterns, six well-delineated clusters were discriminated that, with some exceptions, correlated well with the different groups phenotypically found. Only two strains showed unique protein patterns without meaningful homology to any of the reference strains used for this study and remained unidentified.

Cluster Analysis↗

PubMed1985

Comparison of the exoproducts of gram-negative bacteria by SDS-Page.

The protein exoproducts released during exponential growth of Gram-negative bacteria were analysed and compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-Page). The following bacterial strains were tested: Escherichia coli, Klebsiella pneumoniae, Citrobacter freundii, Enterobacter cloacae, Serratia liquefaciens, Serratia rubidaea, Proteus mirabilis, Proteus vulgaris, Salmonella minnesota, Pseudomonas aeruginosa and Pseudomonas fluorescens. It is demonstrated by SDS-Page that members of one species show identical protein pattern, whereas different species show besides comparable protein bands a species characteristic pattern. All members of Enterobacteriaceae were shown to release proteins whose molecular weights fell into the following size regions: Each strain was shown to synthesize a polypeptide of molecular weight 34,000 and one or more polypeptides within the molecular weight range 25,000-29,000. This profile was shown to be clearly different from that of Pseudomonas strains where 20 or more distinct polypeptides ranging from 12,500 to 160,000 Mr were detectable.

Bacterial Proteins↗

PubMedDate not supplied

[Use of SDS and saponin detergents for the demonstration and purification of (Na+K+)ATPase in sheep kidney].

Incubation of membrane fragments from sheep kidney outer medulla in detergents such as SDS or saponin revealed increased specific (NA+) ATPase activity. Measurement of enzyme phosphorylation from [gamma 32P] ATP showed irreversible alteration of EP to (Na+) ATPase activity ratio when SDS is used as unmasking agent. These results were found with unmasked and purified enzyme.

Adenosine Triphosphate↗

PubMed1998

Fluorimetric and solubility studies of nadolol and atenolol in SDS micelles.

The effect of sodium dodecyl sulfate (SDS) micelles on the spectrofluorimetric intensities and on the solubility of two beta-blockers (atenolol and nadolol) were studied at 25.0 +/- 0.1 degrees C and I = 0.1 M NaCl. From the dependence of these physical properties on SDS concentration it was possible to calculate the binding constants drug-micelle, and it was found that both techniques yield similar results for the binding constants, and that are in agreement with those calculated from the effect of micelles on the apparent acidity constants of the beta-blockers.

Adrenergic beta-Antagonists↗

PubMed2003

Raman spectroscopic investigation of the single-monolayer Langmuir-Blodgett film of C16NaphOH and C10AzoNaphC4N-SDS.

Raman spectra were measured for Langmuir-Blodgett (LB) films of C(16)NaphOH and C(10)AzoNaphC(4)N-SDS on Calcium Fluorite substrate for the first time. In order to find out favorable excitation condition, Raman spectra of the single and multi-monolayer LB films excited at different lines at 244, 514, 633 and 778 nm are recorded and compared in the present study. Raman spectrum of the monolayer LB film of C(16)NaphOH excited by 244 nm demonstrate that excellent signal to noise is achieved even for one monolayer LB film with an extremely short integrating time as 60 s because of being resonantly enhanced, while no meaningful spectra were recorded under the same condition for the monolayer LB film of C(10)AzoNaphC(4)N-SDS because of burning. Using a HeNe 633 nm excitation the problem with strong substrate fluorescence was partially solved, since under these conditions this fluorescence is mainly outside the fingerprint region of the LB film molecules (1000-2000 cm(-1)). Therefore by using the HeNe laser excitation, Raman spectra with high signal to noise ratio of LB films of C(16)NaphOH were collected and shown in this paper. These findings stress again the necessity to define an appropriate Raman system for this special application of LB film diagnosis.

Alkanes↗

PubMed1996

Electrophoretic differentiation of soluble egg antigens from Schistosoma mansoni isolates using SDS-PAGE.

In order to detect intraspecies differences among isolates of Schistosoma mansoni from different geographical regions, soluble egg antigens (SEAs) of three isolates were characterized using SDS-PAGE. SEAs were prepared from eggs of a South African (SA), Eastern Caprivian (EC) and Puerto Rican (PR) isolate, separated by SDS-PAGE, stained and the molecular masses of constituent polypeptides determined. Protein profiles differed for all three isolates with the number of polypeptides varying from 25 for the PR isolate to 27 for the EC and SA isolates. Molecular masses of the polypeptides fall within the range of 12.0 kDa to 97.9 kDa. The EC and SA isolates shared ten common polypeptides, the PR and SA isolates five and the PR and EC isolates two. Only a 30.8 kDa and a 12.0 kDa polypeptide were common to all three isolates. These differences may, to some extent, account for regional variations in the morbidity of intestinal schistosomiasis reported for different geographical isolates of S. mansoni.

Animals↗

PubMed1994

Identification of outbreak-associated and other strains of Clostridium difficile by numerical analysis of SDS-PAGE protein patterns.

Seventy-three cultures of Clostridium difficile isolated both during, and in the period immediately following, an outbreak of infection in a group of three hospitals, were characterized by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell proteins. Each protein pattern was characterized by the presence of one or two dense bands which were highly reproducible. The protein patterns were used as the basis for a numerical analysis which divided the strains into five phenons (electrophoretic or EP types). The majority, 60 of the 73 cultures, belonged to a single phenon which included strains from both patients and the environment. We conclude that high-resolution SDS-PAGE of proteins provides an effective method for typing C. difficile and therefore for tracing the possible spread of epidemic strains in hospitals and other institutions, thereby allowing a better understanding of the epidemiology of the organism.

Adult↗

PubMed2006

Integrated preconcentration SDS-PAGE of proteins in microchips using photopatterned cross-linked polyacrylamide gels.

The potential of integration of functions in microfluidic chips is demonstrated by implementing on-chip preconcentration of proteins prior to on-chip protein sizing by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Two polymeric elements-a thin (approximately 50 microm) size exclusion membrane for preconcentration and a longer (approximately cm) porous monolith for protein sizing-were fabricated in situ using photopolymerization. Contiguous placement of the two polymeric elements in the channels of a microchip enabled simple and zero dead volume integration of the preconcentration with SDS-PAGE. The size exclusion membrane was polymerized in the injection channel using a shaped laser beam, and the sizing monolith was cast by photolithography using a mask and UV lamp. Proteins injected electrophoretically were trapped on the upstream side of the size exclusion membrane (MW cutoff approximately 10 kDa) and eluted off the membrane by reversing the electric field. Subsequently, the concentrated proteins were separated in a cross-linked polyacrylamide monolith that was patterned contiguous to the size exclusion membrane. The extent of protein preconcentration is easily tuned by varying the voltage during injection or by controlling the sample volume loaded. Electric fields applied across the nanoporous membrane resulted in a concentration polarization effect evidenced by decreasing current over time and irreproducible migration of proteins during sizing. To minimize the concentration polarization effect, sieving gels were polymerized only on the separation side of the membrane, and an alternate electrical current path was employed, bypassing the membrane, for most of the elution and separation steps. Electrophoretically sweeping a fixed sample volume against the membrane yields preconcentration factors that are independent of protein mobility. The volume sweeping method also avoids biased protein loading from concentration polarization and sample matrix variations. Mobilities of the concentrated proteins were log-linear with respect to molecular weight, demonstrating the suitability of this approach for protein sizing. Proteins were concentrated rapidly (<5 min) over 1000-fold followed by high-resolution separation in the sieving monolith. Proteins with concentrations as low as 50 fM were detectable with 30 min of preconcentration time. The integrated preconcentration-sizing approach facilitates analysis of low-abundant proteins that cannot be otherwise detected. Moreover, the integrated preconcentration-analysis approach employing in situ formation of photopatterned polymeric elements provides a generic, inexpensive, and versatile method to integrate functions at chip level and can be extended to lowering of detection limits for other applications such as DNA analysis and clinical diagnostics.

Acrylic Resins↗

PubMed1996

Carbon monoxide dehydrogenase from Clostridium thermoaceticum: quaternary structure, stoichiometry of its SDS-induced dissociation, and characterization of the faster-migrating form.

The molecular mass (M(r)) of the nickel- and iron-sulfur-containing enzyme CO dehydrogenase from Clostridium thermoaceticum was determined by sedimentation equilibrium ultracentrifugation to be 300,000 +/- 30,000 Da. Since the enzyme is known to contain equal numbers of two types of subunits (M(r) = 82,000 Da for alpha and 73,000 Da for beta), this indicates an alpha 2 beta 2 quaternary structure. The enzyme was previously thought to have an alpha 3 beta 3 structure because it migrates through calibrated size-exclusion chromatographic columns with an apparent M(r) of about 420,000 Da. The disproportionately fast migration rate suggests that the enzyme is nonspherical. SDS induces the dissociation of an alpha subunit, yielding a stable species called FM-CODH. FM-CODH had a molecular mass of 210,000 +/- 30,000 Da, indicating an alpha 1 beta 2 structure. It contained 2.1 +/- 0.3 Ni and 16 +/- 3 Fe per alpha 1 beta 2, exhibited S-->Fe charge-transfer transitions typical of Fe-S proteins, and afforded the gav = 1.82, 1.86, and 1.94 EPR signals. Quantitation of the 1.82 and (1.94 +/- 1.86) signals afforded 0.35 and 1.9 spin/alpha 1 beta 2, respectively. FM-CODH samples exhibited CO oxidation activity, but little CO/acetyl-CoA exchange activity. Some FM-CODH samples exhibited CO oxidation activities as high as native enzyme. These results, along with the quantified spin intensities of the EPR signals, indicate that FM-CODH contains the B- and C-clusters and suggest that these clusters are located in the beta subunit. The alpha subunit that dissociated during formation of FM-CODH is not required for CO oxidation activity. FM-CODH is either devoid of A-clusters, or if such clusters are present, they have lost their ability to exhibit substantial NiFeC signals and CO/acetyl-CoA exchange activity. Incubating FM-CODH and alpha yielded a species that migrated through polyacrylamide gels at the same rate as native enzyme, and had a molecular mass indicating an alpha 2 beta 2 structure. Thus, the SDS-induced dissociation of the enzyme appears to be reversible.

Aldehyde Oxidoreductases↗