Continuous Flow Electrophoresis Research Articles

Preparation of Highly Enriched ER Membranes Using Free-Flow Electrophoresis.

Free-flow electrophoresis (FFE) is a technique for separation of proteins, peptides, organelles, and cells. With zone electrophoresis (ZE-FFE), organelles are separated according to surface charge. The ER is the only remaining major cellular compartment in Arabidopsis not to have been isolated using density centrifugation, immune-isolation, or any other method previously applied to purification of plant membranes. By using continuous-flow electrophoresis, ER vesicles of similar surface charge, which may have been fragmented during cell lysis, can be focused. A large portion of these vesicles are of sufficiently different surface charge that separation from the majority of Golgi and other contaminants is possible. Here we adapt an earlier ZE-FFE Golgi isolation protocol for the isolation of highly pure ER vesicles and for tracking the migration of peripheral ER vesicles. Isolating ER vesicles of homogeneous surface charge allows multi-omic analyses to be performed on the ER. This facilitates investigations into structure-function relationships within the ER.

Circular Geometry in Molecular Stream Separation to Facilitate Nonorthogonal Field-to-Flow Orientation.

Molecular stream separation (MSS) is a promising complement for continuous-flow synthesis. MSS is driven by forces exerted on molecules by a field applied at an angle to the stream-carrying flow. MSS has only been performed with a 90° field-to-flow angle because of a rectangular geometry of canonic MSS; the second-order rotational symmetry of a rectangle prevents any other angle. Here, we propose a noncanonic circular geometry for MSS, which better aligns with the polar nature of MSS and allows changing the field-to-flow. We conducted in silico and experimental studies of circular geometry for continuous-flow electrophoresis (CFE, an MSS method). We proved two advantages of circular CFE over its rectangular counterpart. First, circular CFE can support better flow and electric-field uniformity than rectangular CFE. Second, the nonorthogonal field-to-flow orientation, achievable in circular CFE, can result in a higher stream resolution than the orthogonal one. Considering that circular CFE devices are not more complex in fabrication than rectangular ones, we foresee that circular CFE will serve as a new standard and a testbed for the investigation and creation of new CFE modalities.

Visualization of Streams of Small Organic Molecules in Continuous-Flow Electrophoresis.

Continuous-flow electrophoresis (CFE) separates a stream of a multicomponent mixture into multiple streams of individual components inside a thin rectangular chamber. CFE will be able to benefit flow chemistry when it is both compatible with nonaqueous solvents utilized in organic synthesis and capable of generically detecting streams of small organic molecules. While stable nonaqueous CFE has been demonstrated, generically detecting molecular streams has not been achieved yet. Here we propose a general approach for molecular stream visualization in CFE via analyte-caused obstruction of excitation of a fluorescent layer underneath the separation chamber-fluorescent sublayer-based visualization (FSV). The concept of FSC-based visualization has been adapted from visualization of small organic molecules on fluorescent plates in thin-layer chromatography. We designed and fabricated a CFE device with one side made of quartz and another side made of UV-absorbing visibly fluorescent, chemically inert, machinable plastic. This device was demonstrated to support nonaqueous CFE of small organic molecules and quantitative detection of their streams in real-time with a limit of detection below 100 μM. Thus, CFE may satisfy conditions required for its seamless integration with continuous flow organic synthesis in flow chemistry.

All-in-one microfluidic device for on-site diagnosis of pathogens based on an integrated continuous flow PCR and electrophoresis biochip.

Current continuous flow polymerase chain reaction (CF-PCR) microfluidic chips require external precision syringe pumps and off-line methods (e.g., electrophoresis and hybridization) to detect PCR products, resulting in complex operations and possible cross-contamination and consequently CF-PCR is still confined to laboratories. Herein, a portable all-in-one microfluidic device is fabricated for rapid diagnosis of pathogens based on an integrated CF-PCR and electrophoresis biochip. A new method was proposed for automatic sample injection into the chip which can substitute the costly external precision syringe pump. It not only achieves rapid DNA amplification and on-site PCR product detection, but also realizes automatic sample injection. As an application, three periodontal pathogens (e.g., Porphyromonas gingivalis, Treponema denticola and Tannerela forsythia) were successfully amplified in the device. Treponema denticola was amplified in as short as 2'31'', and detection of PCR products was completed within 3'43''. The minimum number of bacteria that can be amplified was 125 cfu per μl. The all-in-one device has the potential to be applied in point-of-care nucleic acid testing for diseases.

Non-aqueous continuous-flow electrophoresis (NACFE): potential separation complement for continuous-flow organic synthesis

We introduce non-aqueous continuous-flow electrophoresis (NACFE) in which the electrolyte is a solution of an organic salt in an aprotic organic solvent. NACFE can maintain steady-state separation of multiple hydrophobic organic species into individual molecular streams. It is a potential separation complement for continuous-flow organic synthesis. This proof-of-concept work will serve as a justification for efforts towards making NACFE a practical tool in flow chemistry.

Preparation of Highly Enriched ER Membranes Using Free-Flow Electrophoresis.

Free-flow electrophoresis (FFE) is a technique for separation of proteins, peptides, organelles, and cells. With zone electrophoresis (ZE-FFE), organelles are separated according to surface charge. The ER is the only remaining major cellular compartment in Arabidopsis not to have been isolated using density centrifugation, immune-isolation, or any other method previously applied to purification of plant membranes. By using continuous-flow electrophoresis ER vesicles of similar surface charge, which may have been fragmented during cell lysis, can be focused. A large portion of these vesicles are of sufficiently different surface charge that separation from the majority of Golgi and other contaminants is possible. Here we adapt an earlier ZE-FFE Golgi isolation protocol for the isolation of highly pure ER vesicles and for tracking the migration of peripheral ER vesicles. Isolating ER vesicles of homogenous surface charge allows multi-'omic analyses to be performed on the ER. This facilitates investigations into structure-function relationships within the ER.

Continuous-Flow Electrophoresis of DNA and Proteins in a Two-Dimensional Capillary-Well Sieve.

Continuous-flow electrophoresis of macromolecules is demonstrated using an integrated capillary-well sieve arranged into a two-dimensional anisotropic array on silicon. The periodic array features thousands of entropic barriers, each resulting from an abrupt interface between a 2 μm deep well (channel) and a 70 nm capillary. These entropic barriers owing to two-dimensional confinement within the capillaries are vastly steep in relation to those arising from slits featuring one-dimensional confinement. Thus, the sieving mechanisms can sustain relatively large electric field strengths over a relatively small array area. The sieve rapidly sorts anionic macromolecules, including DNA chains and proteins in native or denatured states, into distinct trajectories according to size or charge under electric field vectors orthogonally applied. The baseline separation is achieved in less than 1 min within a horizontal migration length of ∼1.5 mm. The capillaries are self-enclosed conduits in cylindrical profile featuring a uniform diameter and realized through an approach that avoids advanced patterning techniques. The approach exploits a thermal reflow of a layer of doped glass for shape transformation into cylindrical capillaries and for controllably shrinking the capillary diameter. Lastly, atomic layer deposition of alumina is introduced for the first time to fine-tune the capillary diameter as well as to neutralize the surface charge, thereby suppressing undesired electroosmotic flows.

High-Speed, Comprehensive, Two Dimensional Separations of Peptides and Small Molecule Biological Amines Using Capillary Electrophoresis Coupled with Micro Free Flow Electrophoresis.

Two-dimensional (2D) separations are able to generate significantly higher peak capacities than their one-dimensional counterparts. Unfortunately, current hyphenated 2D separations are limited by the speed of the second dimension separation and the consequent loss of peak capacity due to under sampling of peaks as they elute from the first dimension separation. Continuous micro free flow electrophoresis (μFFE) separations eliminate under sampling as a limitation when incorporated as the second dimension of a 2D separation. In the current manuscript we describe the first coupling of capillary electrophoresis (CE) with μFFE to perform 2D CE × μFFE separations. The CE separation capillary was directly inserted into the μFFE separation channel using an edge on interface. Analyte peaks streamed directly into the μFFE separation channel as they migrated off the CE capillary. No complicated injection, valving, or voltage changes were necessary to couple the two separation modes. 2D CE × μFFE generated an ideal peak capacity of 2 592 in a 9 min separation of fluorescently labeled peptides (7.6 min separation window, 342 peaks/min). Data points were recorded every 250-500 ms (>8 data points/peak), effectively eliminating under sampling as a source of band broadening. CE × μFFE generated an ideal peak capacity of 1885 in a 2.7 min separation of fluorescently labeled small molecule bioamines (1.8 min separation window, 1053 peaks/min). Peaks in the 2D CE × μFFE separation of peptides covered 30% of the available separation space, resulting in a corrected peak capacity of 778 (102 peaks/min). The fractional coverage of the 2D CE × μFFE separation of small molecule bioamines was 20%, resulting in a corrected peak capacity of 377 (209 peaks/min).

Confinement effects on electrohydrodynamics of two-dimensional miscible liquid drops

The effect of domain confinement on the electrohydrodynamics of a two-dimensional miscible drop is investigated analytically, as a prototypical problem to explore confinement effect in two applications; namely, continuous flow electrophoresis and pattern formation in suspension of inhomogeneous colloidal dispersions. It is shown that the domain confinement may strengthen or weaken the electric field and leads to weakening of the flow field and change of the flow structure. A characteristic function is found to determine the sense of drop elongation and it is shown that below a critical domain size, the necessary condition for elongation in the direction normal to the field will be opposite to that for an unbounded domain.

Asynchronous grid computing for the simulation of the 3D electrophoresis coupled problem

This paper deals with the implementation on the Grid'5000 network, the French grid platform, of parallel simulations for the solution of a 3D coupled boundary value problem. This problem models continuous flow electrophoresis which is governed by Navier Stokes equations coupled with convection-diffusion and potential equations. The coupled boundary value problems are discretised by appropriate schemes involving the convergence of the parallel synchronous and asynchronous Schwarz alternating algorithms. Finally, parallel experiments on grid computing are presented and analyzed.

Electrokinetic behaviour and surface sialic acid status of blood platelets in essential thrombocythaemia (ET).

We have previously reported, for platelets from patients with idiopathic thrombocytopenic purpura, that there was in increased electrophoretic mobility that was related to their increased sialic acid content. Heterogeneity in the circulating platelet pool of patients with essential thrombocythaemia (platelet counts 600-1000 x 10(9)/l) has also been investigated by preparative continuous flow electrophoresis (CFE). The surface charge-dependent separation profiles of all the patients' platelets showed marked anodal shifts compared with the profiles of platelets from age- and sex-matched control subjects separated under identical conditions. The increase above normal in the net electronegativity of the patients' platelets could not be accounted for by differences in surface neuraminidase-labile or total platelet sialic acid content.

Parallel asynchronous iterations for the solution of a 3D continuous flow electrophoresis problem

This paper deals with the parallel numerical solution of a 3D continuous flow electrophoresis problem governed by Navier–Stokes equations coupled with transport and potential equations. For this problem, using the properties of the discrete operators, the convergence of synchronous and asynchronous parallel Schwarz alternating methods is analysed. Finally, parallel solvers are implemented and the results of simulations are given.

Ionic Strength and pH-Induced Changes in the Immunoreactivity of Purified Soybean Glycinin and Its Relation to Protein Molecular Structure

This study examined the immunogenic response of glycinin under varying conditions of pH and ionic strength using enzyme-linked immunosorbent assay. Differential scanning calorimetric (DSC) analysis and Fourier transform infrared spectroscopy (FTIR) were used to investigate the conformational changes induced as a result of these conditions, and the correlation with the changes observed in glycinin immunoreactivity were determined. A highly purified glycinin obtained by isoelectric precipitation followed by native preparative continuous flow electrophoresis was used for these studies. Purity was confirmed by two-dimensional-polyacrylamide gel electrophoresis and mass spectroscopy. DSC and FTIR results suggest that glycinin immunoreactivity is affected by changes in the tertiary and secondary packing of the protein, when flexibility, stability, and accessibility of certain substructures are modified. Aggregation and/or increased compactness of glycinin subcomponents could have potentially prevented epitopes from reacting with the IgG antibodies.

Parallel numerical simulation for the coupled problem of continuous flow electrophoresis

AbstractThe performance of parallel subdomain method with overlapping is analysed in the case of the 3D coupled boundary‐value problem of continuous flow electrophoresis which is governed by Navier–Stokes equations coupled with convection–diffusion and potential equations. Convergence of parallel synchronous and asynchronous iterative algorithms is studied. Comparison between implemented explicit and implicit schemes for the transport equation is made using these algorithms and shows that both methods provide similar results and comparable performances. Copyright © 2007 John Wiley & Sons, Ltd.

A novel 78-kDa fatty acyl-CoA synthetase (ACS1) of Babesia bovis stimulates memory CD4 + T lymphocyte responses in B. bovis-immune cattle

Antigen-specific CD4 + T lymphocyte responses contribute to protective immunity against Babesia bovis, however the antigens that induce these responses remain largely unknown. A proteomic approach was used to identify novel B. bovis antigens recognized by memory CD4 + T cells from immune cattle. Fractions obtained from merozoites separated by continuous-flow electrophoresis (CFE) that contained proteins ranging from 20 to 83 kDa were previously shown to stimulate memory CD4 + lymphocyte responses in B. bovis-immune cattle. Expression library screening with rabbit antiserum raised against an immunostimulatory CFE fraction identified a clone encoding a predicted 78 kDa protein. BLAST analysis revealed sequence identity of this B. bovis protein with Plasmodium falciparum fatty acyl coenzyme A synthetase (ACS) family members (PfACS1–PfACS11), and the protein was designated B. bovis acyl-CoA synthetase 1 (ACS1). Southern blot analysis indicated that B. bovis ACS1 is encoded by a single gene, although BLAST analysis of the preliminary B. bovis genome sequence identified two additional family members, ACS2 and ACS3. Peripheral blood lymphocytes and CD4 + T cell lines from B. bovis-immune cattle proliferated significantly against recombinant ACS1 protein, consistent with its predicted involvement in protective immunity. However, immune sera from cattle recovered from B. bovis infection did not react with ACS1, indicating that epitopes may be conformationally dependent.

Pressure injection in continuous sample flow electrophoresis microchips

Pressure pulse injection is proposed as an alternative technique to electrokinetic injection for use in continuous sample flow electrophoresis microchips. During chip operation, sample and buffer solutions are electrokinetically driven near to the entrance of the separation channel in a continuous way. A short sample plug is then injected in the separation channel by a pressure pulse of a few hundred of milliseconds, by mechanically deflecting a soft polymer membrane placed on a dedicated chip reservoir. Our pressure pulse injection technique does not depend on the charge of the analytes and, hence, is characterized by a strongly reduced bias compared to a classical gated flow injection sequence.

Identification of Ehrlichia ruminantium (Gardel strain) IFN-γ inducing proteins after vaccination with a killed vaccine

IFN-γ is considered as a key factor in protection against heartwater of ruminants, caused by the obligate intracellular bacterium Ehrlichia ruminantium. In this study, a better definition of the molecular masses of IFN-γ inducing proteins of the Gardel strain of E. ruminantium was obtained by the use of continuous flow electrophoresis (CFE) and sensitized polyclonal lymphocytes. Out of 15 E. ruminantium CFE fractions tested within the 14–39 kDa region, eight were commonly reacted to by all goats. Interestingly, half of these fractions fall within the 23–29 kDa region, shown previously to contain polymorphic B-cell epitopes. Thus, the results suggest that this region also contains T-cell epitopes potentially involved in protection. Also, several proteins were found to be more immunogenic than the serologically immunodominant MAP1 protein. Finally, high activity within the 15–19 kDa region was observed, which confirms previous work done with CD4+ T-cell lines obtained from cattle immunized with a South African strain of E. ruminantium. The proteins falling within the molecular weight ranges defined in this study may have potential as vaccine antigens.

Free-flow electrophoresis in a microfabricated chamber with a micromodule fraction separator: Continuous separation of proteins

Continuous free flow electrophoresis of proteins was carried out in a microfabricated free flow electrophoresis (mFFE) module with the 30-μm thick slit of the separation. The newly developed micromodule fraction separator (MFS) was attached to the down-stream end site of the separation chamber of mFFE. By using the MFS, electrolyte solution from the separation chamber was introduced to the peristaltic pump without disturbing the electrolyte solution flow at the bottom side of the chamber. The separation of protein mixture samples was achieved by a hydroxypropylmethylcellulose pretreatment coating of the separation chamber. The pretreatment of the sample chamber effectively suppressed electroosmotic flow. All fractionated samples were collected using the MFS after continuous elecrophoresis and analyzed by reversed-phase HPLC. From the results of HPLC analyses none of the cytochrome c fractions at the other ports revealed cross talk phenomena at adjacent ports. A similar result occurred for the myoglobin. This means that these proteins were completely separated from each other by continuous mFFE, and the MFS functioned efficiently during continuous electrophoresis.

Cowdria ruminantium antigens of around 15 kDa are potent inducers of IFN-gamma.

Cellular responses induced in two Creole goats by vaccination with killed Cowdria ruminantium (Cowdria) were confirmed by IFN-gamma production and interleukin-2 receptor (IL-2R) expression. Both CD4+ and CD8+ but not WC1+ T cells showed a substantial increase in cell surface expression of IL-2R molecules in response to whole Cowdria lysate. Cowdria (Welgevonden strain) proteins were fractionated using continuous-flow electrophoresis and tested for their ability to induce IFN-gamma production by PBMC collected three weeks after the first inoculation and one week after the booster injection. Pooled fractions of around 15, 22, and 24 kDa were found to induce significant IFN-gamma production in both vaccinated animals on one of the two occasions. Antigens of around 15 kDa induced substantially higher IFN-gamma production than any other fractions in both animals. These pilot experiments pave the way towards the identification of proteins/genes that have potential for the development of a recombinant vaccine against heartwater.

Optimization of preparative electrophoretic chiral separation of ritalin enantiomers

Continuous free flow electrophoresis (CFFE) was applied to the preparative chiral separation of ritalin enantiomers. Sulfated β-cyclodextrin (sβ-CD) was used as the chiral additive. Liquid chromatography–mass spectrometry (LC–MS) experiments were applied to study the time averaged concentration of sβ-CD in the separation chamber. The distribution of sβ-CD in the separation chamber greatly influenced resolution and the angle of deflection. To optimize the separation, several parameters (methanol, concentration of sβ-CD in the cathodic wash and in the separation buffer, and the introduction of a low conductivity zone) were investigated. The dependence of the resolution and deflection angles of ritalin enantiomers on the concentration of sβ-CD in both the separation buffer and in the cathode wash solution appeared to be non-linear. Under close to optimal conditions, resolution of ritalin enantiomers was about 0.8 with an average processing rate of 0.5 mg/h. Overall, the enantiomeric purity of the individual isomers was ∼83%; however, of the 20 vials containing ritalin, the presence of both enantiomers was only detected in three vials.