SDS-PAGE under focusing conditions: an electrokinetic transport phenomenon based on charge compensation

A novel method is reported for mass separation of proteins, based on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Unlike conventional SDS-PAGE, in which separation by mass of SDS-laden polypeptide chains is obtained in constant concentration or porosity gradient gels, the present method, called "SDS-PAGE focusing", exploits a "steady-state" process by which the SDS-protein micelles are driven to stationary zones along the migration path against a gradient of positive charges affixed to the neutral polyacrylamide matrix. As the total negative surface charge of such complexes matches the surrounding charge density of the matrix, the SDS-protein complex stops migrating and remains stationary, as typical of steady-state separation techniques. As a result of this mechanism, the proteins are separated in an unorthodox way, with the smaller proteins/peptides staying closer to the application point and larger proteins migrating further down toward the anodic gel end. This results in a positive slope of the Mr vs migration plot, vs a negative slope in conventional SDS-PAGE. Moreover, such a plot is linear (by design), whereas in standard SDS-PAGE it is semi- or even double logarithmic. Particularly advantageous appears the ability of the present method to fine-tune the separation of small-size fragments and tryptic digests, where conventional SDS-PAGE usually fails. Additionally, by exploiting constant plateaus of charges, rather than gradients, it is possible to amplify the separation between species having closely spaced Mr values, down to a limit of approximately 150 Da. This increases the resolution by at least 1 order of magnitude as compared with standard SDS-PAGE, where for a proper separation of two adjacent species, an Mr increment of approximately 3000 Da is needed.     

Polymeric micelles comprising stearic acid-grafted polyethyleneimine as nonviral gene carriers

Non-viral vectors composed of biodegradable polymers or lipids have been considered as a safer alternative for gene carriers over viral vectors. Among some of the cationic polymers, polyethyleneimine (PEI) possess high pH-buffering capacity that can provide protection to nucleotides from acidic degradation and promotes endosomal and lysosomal release. However, it has been reported that cytotoxicity of PEI depends on the molecular weight of the polymer. Hence modifications of PEI structure for clinical application have been developed in order to reduce the cytotoxicity, or improve the insufficient transfection efficiency of lower molecular weight PEI. In this study, 10 k PEI was modified by grafting stearic acid (SA) and formulated to polymer micelles with positive surface charge and evaluated for pDNA delivery. The amine group on PEI was crosslinked with the carboxylic group of stearic acid by 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDC) as linker. PEI-SA micelles were then prepared using oil in water (o/w) solvent evaporation method. The success of PEI-SA conjugation structure was confirmed with 1H NMR. The average diameter and zeta potential determined by photon correlation spectroscopy was 149.6 +/- 1.2 nm and 64.1 +/- 1.5 mV, respectively. These self-assemble positive charge micelles showed effective binding to pDNA for transfection. PEI-SA micelles exhibited lower cytotoxicity compared to that of PEI only, while flow cytometry analysis revealed PEI-SA/pEGFP complex provided 62% high EGFP expression. Luciferase activity also showed high transfection efficiency of PEI-SA micelles for weight ratio above 4.5 that was comparable to PEI only. These results demonstrated that stearic acid grafted PEI micelles can provide high transfection efficiency comparable to unmodified PEI, and exhibit low cytotoxicity. Stearic acid grafted PEI micelles can be promising polymer carriers in genetic therapy.     

Adsorption of conditioning polymers on solid substrates with different charge density

The adsorption processes of polymers that belong to two different families (neutral hydrophilic polymers and cationic polysaccharide polymers) onto solid surfaces with different charge density have been studied using dissipative quartz crystal microbalance (D-QCM) and ellipsometry. The polymers studied are very frequently used in the cosmetic industry as conditioning agents. The adsorption kinetics of the polymers involves at least two steps. The total adsorbed amount depends on the charge density of the surface for both types of polymers. The comparison of the adsorbed mass on each layer obtained from D-QCM and from ellipsometry has allowed calculating the water content of the layers that reaches high values for the polymers studied. The analysis of D-QCM results also provided information about the shear modulus of the layers, whose values have been found to be typical of a rubber-like polymer system. The main driving force of the adsorption was found to be the energy of the interactions between chains and surface.     

Staining method for protein analysis by capillary gel electrophoresis

A novel staining method and the associated fluorescent dye were developed for protein analysis by capillary SDS-PAGE. The method strategy is to synthesize a pseudo-SDS dye and use it to replace some of the SDS in SDS-protein complexes so that the protein can be fluorescently detected. The pseudo-SDS dye consists of a long, straight alkyl chain connected to a negative charged fluorescent head and binds to proteins just as SDS. The number of dye molecules incorporated with a protein depends on the dye concentration relative to SDS in the sample solution, since SDS and dye bind to proteins competitively. In this work, we synthesized a series of pseudo-SDS dyes, and tested their performances for capillary SDS-PAGE. FT-16 (a fluorescein molecule linked with a hexadodecyl group) seemed to be the best among all the dyes tested. Although the numbers of dye molecules bound to proteins (and the fluorescence signals from these protein complexes) were maximized in the absence of SDS, high-quality separations were obtained when co-complexes of SDS-protein-dye were formed. The migration time correlates well with protein size even after some of the SDS in the SDS-protein complexes was replaced by the pseudo-SDS dye. Under optimized experimental conditions and using a laser-induced fluorescence detector, limits of detection of as low as 0.13 ng/mL (bovine serum albumin) and dynamic ranges over 5 orders of magnitude in which fluorescence response is proportional to the square root of analyte concentration were obtained. The method and dye were also tested for separations of real-world samples from E. coli.     

运用一步化体外转录—翻译系统筛查基因突变的新技术

报道了一种筛查基因突变的新技术－ＰＣＲ结合一步化体外转转录－翻译系统。该技术包括：（１）ＰＣＲ扩增靶ＤＮＡ片段，并使ＤＮＡ产物的一端或两端带上Ｔ７Ｔ１序列；（Ｔ１：翻译起始信号，ＣＣＡ ＣＣＡ ＴＧ）；（２）以核的为模板，利用一步化体外转录－翻译系统合成相应肽链；（３）用ＳＤＳ－ＰＡＧＥ及高分辨ｐＨ梯度ＰＡＧＥ（聚丙烯酰胺凝胶电泳）分离分析多肽产物，通过异常电泳图谱而发现基因突变，本文以已知突变的     

Charge re-distribution and ZT in PEDOT-related thermoelectrics

Recently it has been reported that poly(3,4-ethylenedioxythiphene (PEDOT))-related polymers can attain values of ZT comparable to their inorganic counterpart. In particular, it was shown that the thermal conductivity κ could be lowered to a value found in nano-structured semiconductors. In this work we studied the charge transport properties of PEDOT-related polymers and examined how the materials parameters were linked to κ and ZT based on data reported in the open literature. Using a two-region model and taking into account charge migration from the more conducting PEDOT islands to the resistive PSS sheaths in poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate), we were able to demonstrate that ZT in the PSS sheaths could increase by orders of magnitude. Furthermore, ZT could approach a value of 20 or more when σ the bandwidth of the density of states of the transport sites was very small (<3 meV). Outward charge migration from the islands can in theory be enhanced by adding negatively-charged oxygen ions to the sheaths as observed in oxidized Poly(3,4-ethylenedioxythiophene-tosylate). This lowers the barrier height for holes and enhances ZT in the sheaths.     

Effect of molecular architecture of polycarboxylate ethers on plasticizing performance in alkali-activated slag paste

A range of anionic and cationic polycarboxylate ether (PCE) plasticizers with different molecular architectures (molecular weights, side chain lengths, and ratios of side chain density to backbone charge) are synthesized and tested to determine their effects on the rheological properties of fresh alkali-activated slag (AAS) pastes. A higher density of long side chains in the lower molecular weight polymers can provide a noticeable yield stress reduction, indicating a mild increase in workability compared to that of an unmodified AAS paste. It is hypothesized that side chains may have two important roles, i.e., providing steric hindrance to disperse particles after PCE adsorption on a particle surface, and also providing partial protection of the backbone charges against attachment of one PCE molecule to two or more slag particles, which is called bridging. This enhances the likelihood of adsorption on single particles, and thus increases the plasticizing action. A very similar plasticizing mechanism is observed for PCEs with similar structures but differing charge signs (cationic/anionic), which indicates that both anionic and cationic adsorption sites are available on AAS particle surfaces. The measured flow curves of all pastes are well described by the Herschel–Bulkley model with shear thinning behavior.     

Relationship between aggregate and sediment bed properties: Influence of inter-particle adhesion

The influence of the magnitude of inter-particle attraction and adhesion, aggregate size and aggregate structure on the shear and compressive rheological behaviour of flocculated suspensions has been studied. Ninety nanometer diameter silica particles have been flocculated with poly diallyldimethylammonium chloride based cationic polymers of molecular weight approximately 100–300 kDa and 10–100% cationic charge. The amount of charge on the polymer influences the adsorption conformation of the polymer which influences the dominant mechanism for inter-particle attraction. Low charge density results in primarily bridging flocculation while high charge density results in primarily charge patch attraction. Although the bridging adhesion is generally lower in magnitude than for the charge patch, the bridging flocculation typically produces large aggregates while the charge patch mechanism results in smaller aggregates due to the influence of shear during aggregation. The optimum dose for aggregation (supernatant clarity) of each type of polymer corresponds well with the polymer concentration which produces the maximum adhesive force measured with AFM. The influence of aggregate size on the compressive and shear yield stresses is weak, but the compressive and shear yield stresses increase linearly with increasing adhesion force.     

LHRH—PEG—PHIS胶束的制备与性质研究

通过化学法制备得到pH响应的、促黄体激素释放激素介导的聚乙二醇一聚组氨酸（LHRH—PEG—PHIS）纳米胶束,采用傅立叶红外光谱（FTIR）及核磁共振氢谱（IHNMR）对LHRH—PEG—PHIS聚合物结构进行表征,利用动态光散射法（DLS）、荧光分光光度计、透射电镜（TEM）等对LHRH—PEG—PHIS胶束的性质进行了一系列研究,结果表明．选择性溶剂的种类、选择性溶剂与共同溶剂之间的质量比及聚合物初始浓度等因素都会对胶束的形态产生影响。制备得到的胶柬约90nm,临界胶柬浓度为10μg／mL,且胶束表面在pH7．4时呈现负电荷,而到肿瘤外呈现正电荷。由于质子化的组氨酸链段间静电排斥作用,在pH5．0时,LHRH—PEG—PHIS胶柬的粒径和zeta电位均比pH7．4时大。细胞毒性研究表明该胶束材料无毒性．因此,LHRH—PEG—PHIS纳米胶柬可望成为抗肿瘤药物载体。     

Migration behavior of cationic solutes in micellar electrokinetic capillary chromatography.

A phenomenological approach is presented to describe the migration of cationic solutes in micellar electrokinetic capillary chromatography (MECC). The migration behavior of an organic base is complicated by the presence of an acid-base equilibrium, the ion-pairing formation between the conjugated acid of the base and the monomer surfactants, and the interactions of both the base and its conjugated acid with the micellar pseudophase. An equation was derived that allows the calculation of the migration factor of a cationic solute in MECC with anionic micelles. Two limiting cases were considered: first the cationic solute completely associates with the anionic surfactant (ion-pair formation constant, KIP, approaches infinity), and therefore there is no free charged species in the solution; second, the KIP = 0 and the free conjugated acid, BH+ migrates in the aqueous bulk solvent at its own electrophoretic velocity. An estimate for the ion-pair formation constant between cationic solutes and free surfactant can be obtained by using the model.     

Enhanced separation of membranes during free flow zonal electrophoresis in plants

Free flow zonal electrophoresis (FFZE) is a versatile technique that allows for the separation of cells, organelles, membranes, and proteins based on net surface charge during laminar flow through a thin aqueous layer. We have been optimizing the FFZE technique to enhance separation of plant vacuolar membranes (tonoplast) from other endomembranes to pursue a directed proteomics approach to identify novel tonoplast transporters. Addition of ATP to a mixture of endomembranes selectively enhanced electrophoretic mobility of acidic vesicular compartments during FFZE toward the positive electrode. This has been attributed to activation of the V-ATPase generating a more negative membrane potential outside the vesicles, resulting in enhanced migration of acidic vesicles, including tonoplast, to the anode (Morré, D. J.; Lawrence, J.; Safranski, K.; Hammond, T.; Morré, D. M. J. Chromatogr., A 1994, 668, 201-213). We confirm that ATP does induce a redistribution of membranes during FFZE of microsomal membranes isolated from several plant species, including Arabidopsis thaliana, Thellungiella halophila, Mesembryanthemum crystallinum, and Ananas comosus. However, we demonstrate, using V-ATPase-specific inhibitors, nonhydrolyzable ATP analogs, and ionophores to dissipate membrane potential, that the ATP-dependent migrational shift of membranes under FFZE is not due to activation of the V-ATPase. Addition of EDTA to chelate Mg2+, leading to the production of the tetravalent anionic form of ATP, resulted in a further enhancement of membrane migration toward the anode, and manipulation of cell surface charge by addition of polycations also influenced the ATP-dependent migration of membranes. We propose that ATP enhances the mobility of endomembranes by screening positive surface charges on the membrane surface.     

Enrichment of Cations via Bipolar Electrode Focusing

We have previously demonstrated up to 5 × 10(5)-fold enrichment of anionic analytes in a microchannel using a technique called bipolar electrode focusing (BEF). Here, we demonstrate that BEF can also be used to enrich a cationic fluorescent tracer. The important point is that chemical modification of the microchannel walls enables reversal of the electroosmotic flow (EOF), enabling cations, instead of anions, to be enriched via an electric field gradient focusing mechanism. Reversal of the EOF has significant consequences on the formation and shape of the region of the buffer solution depleted of charge carriers (depletion zone). Electric field measurements and numerical simulations are used to elucidate the factors influencing the depletion zone. This information is used to understand and control the location and shape of the depletion zone, which in turn influences the stability and concentration of the enriched band.     

纤维素酶调控预处理对MFC膜包装性能的影响

目的提高纤维素酶预处理效率,改善微纤化纤维素(MFC)薄膜的性能。方法以漂白桉木硫酸盐浆为原料,通过分别添加阳离子聚合物聚二甲基二烯丙基氯化铵(PDADMAC)和聚丙烯酰胺(CPAM)来调控纤维素酶的酶活性(Cx酶活性),并对比分析阳离子种类和添加量对MFC薄膜拉伸性能、透氧性能、透光性能的影响。结果高电荷量、低分子量的PDADMAC对酶活性的提升作用较大,提高了酶的预处理效率;高分子量、高电荷量的CPAM虽然降低了酶活性,但在一定程度上有效控制了酶在无定形区的过度水解,有利于薄膜力学性能、阻隔性能的提高。结论添加阳离子聚合物能够提高纤维素酶的Cx酶活性,进而提高纤维素酶对漂白桉木硫酸盐浆的预处理效率,最终获得性能优良的MFC薄膜产品。     

A study of multicompartment polymeric micelles

A series of novel polymerizable hydrocarbon (H) and fluorocarbon (F) cationic acrylamido surfactants were prepared, characterized and used as comonomers for the synthesis of multicompartment polymeric micelles. The nature of the amido group of the surfactant, either N-monosubstituted (CONRH) or disubstituted (CONRC₂H₅) is one of the key parameters governing their solution behavior. Thus, micellar growth for pure F-surfactants and miscibility/incompatibility for mixed systems depend on the possibility of H-bond formation. Radical polymerization of acrylamide in aqueous solution in the presence of demixing hydrocarbon and fluorocarbon polymerizable micelles leads to water-soluble polymers containing well segregated hydrocarbon and fluorocarbon domains as confirmed by fluorescence spectroscopy.     

Characterizing poly(epsilon-caprolactone)-b-chitooligosaccharide-b-poly(ethylene glycol) (PCP) copolymer micelles for doxorubicin (DOX) delivery: effects of crosslinked of amine groups

New amine-groups containing tri-block copolymers and micelles that consisting of poly(epsilon-caprolactone)-b-chitooligosaccharide-b-poly(ethylene glycol) (PCL-b-COS-b-PEG, PCP), were synthesized, characterized, and evaluated for delivering doxorubicin (DOX) with or without crosslinked amine groups by genipin. The characteristics of the PCP copolymers of Fourier-transform infrared spectrometry (FT-IR) verify the amine and ester groups of the COS and the PCL of the copolymers, respectively. 1H nuclear magnetic resonance (1H NMR) spectra verify the structures of the PCP copolymers consisting two PCL and PEG polymers reacted onto the COS block. In addition, gel permeation chromatography (GPC) determines the number average molecular weight of the tri-block copolymers (Mn) of approximately 11340 Da/mole. The PCP copolymers can self-assemble to form polymeric micelles at the critical micelle concentration (CMC) of 1.0 microM as determined by the UV-VIS absorption spectra. The mean diameter of the PCP micelles is 90 nm, as determined using a dynamic light-scattering (DLS) analyzer. Moreover, the zeta potentials of PCP micelles change from neutral to cationic state when pH of suspension mediums varied from 7.4 to 3.0. For evaluating delivery characteristics of hydrophobic DOX, it was loaded into PCP micelles with or without crosslinked by genipin. The burst release and release period of DOX for the crosslinked micelles are significantly reduced (P < 0.003, n = 3, for pH = 7.4) and sustained (e.g., 8 days), respectively, than those non-crosslinked ones (e.g., 4 days). In conclusion, new tri-block amine groups containing PCP copolymers are synthesized that can self-assemble as PCP micelles. After post-crosslinked amine groups of DOX loaded the micelles, they can effectively reduce the burst release and sustain the release of DOX at different pH dissolution mediums. Further applications of PCP copolymers and micelles for drug delivery can be explored in future.     

A Nanofilter Array Chip for Fast Gel-Free Biomolecule Separation

We report here a microfabricated nanofilter array chip that can size-fractionate SDS-protein complexes and small DNA molecules based on the Ogston sieving mechanism. Nanofilter arrays with a gap size of 40-180nm were fabricated and characterized. Complete separation of SDS-protein complexes and small DNA molecules were achieved in several minutes with a separation length of 5mm. The fabrication strategy for the nanofilter array chip allows further increasing of the nanofilter density and decreasing of the nanofilter gap size, leading, in principle, to even faster separation.     

A novel drug and gene co-delivery system based on Poly(epsilon-caprolactone)-Poly(ethylene glycol)-Poly(epsilon-caprolactone) grafted polyethyleneimine micelle

In this paper, we prepared a novel cationic self-assembled micelle from poly(epsilon-caprolactone)-poly(ethyl glycol)-poly(epsilon-caprolactone) grafted polyethyleneimine (PCEC-g-PEI). The PCEC-g-PEI micelles, formed by self-assembly method, had mean particle size of ca. 82 nm and zeta potential of +22.5 mV at 37 degrees C, and could efficiently transfer pGFP into HEK293 cells in vitro. Meanwhile, as a model hydrophobic chemotherapeutic drug, honokiol was loaded into PCEC-g-PEI micelles by direct dissolution method assisted by ultrasonication. The honokiol loaded cationic PCEC-g-PEI micelles could effectively adsorb DNA onto its surface, while it could release honokiol in an extended period in vitro. This study demonstrated a novel DNA and hydrophobic chemotherapeutic drug co-delivery system.     

Predictive study of charge transport in disordered semiconducting polymers

We present a theoretical study of charge transport in disordered semiconducting polymers that relates the charge mobility to the chemical structure and the physical morphology in a novel multiscale approach. Our studies, focusing on poly(9,9-dioctylfluorene) (PFO), show that the charge mobility is dominated by pathways with the highest interchain charge-transfer rates. We also find that disorder is not always detrimental to charge transport. We find good agreement with experimental time-of-flight mobility data in highly aligned PFO films.     

Capillary electrophoresis sodium dodecyl sulfate nongel sieving analysis of a therapeutic recombinant monoclonal antibody: a biotechnology perspective.

With the increasing interest in the therapeutic use of recombinant monoclonal antibodies (rMAbs), a generic analytical approach for the analysis of size-based rMAb variants is desired. Such a method using capillary electrophoresis (CE) with laser-induced fluorescence detection is described. The assay was developed as a replacement for silver-stained SDS-PAGE and was validated according to the guidelines of the International Committee on Harmonization for use in routine lot release testing of a rMAb pharmaceutical. In this assay, the rMAb solution is first derivatized with a neutral fluorophore, e.g., 5-carboxytetramethylrhodamine succinimidyl ester. The labeled sample is then incubated with SDS, and the SDS-protein complexes are then separated by CE using a hydrophilic polymer as a sieving matrix. The precolumn labeling conditions described in this study allowed the detection of rMAb at a low-nanomolar concentration (9 ng/mL), with no apparent loss in resolution or changes to the distribution of rMAb analyte species, when compared to an unlabeled sample. In addition, the traditional practice of heating proteins at elevated temperatures in the presence of SDS to facilitate SDS-protein binding resulted in the generation of significant levels of rMAb fragmentation, and alternative conditions to minimize this artifact are discussed. Illustrations of the uses of this assay in monitoring consistency of bulk manufacture of a protein pharmaceutical, and in providing a size-based separation of product-related variants, as well as nonproduct impurities are shown. In brief, the assay described in this paper demonstrated comparable resolution and sensitivity to silver-stained SDS-PAGE but offered the advantages of enhanced precision and robustness, speed, ease of use, and on-line detection.     

Synthesis and electrostatic nano-assembly of water-soluble polybenzothiadiazole derivatives with long-wavelength emission in the solid states

We have synthesized APBT and APTBT containing benzothiadiazole units by Suzuki cross-coupling reaction with good yield. The polymers showed blue emission colors in aqueous solutions, while long wavelength shift was observed in the solid state due to facilitated exciton migration. APBT and APTBT are water-soluble and highly-fluorescent conjugated polymers with negatively charged sulfonate side chains and thus they can be electrostatically assembled with oppositely charged polyelectrolyte such as cationic polymer, poly(dimethyldiallylammonium chloride) (PDAC) via layer-by-layer (LbL) deposition technique on a glass slide. According to the increased the number of bilayer, we found that the assembled film exhibited larger enhancement of the long wavelength emission relative to the blue emission, due to the increased excition migration.