Development of a novel two-dimensional gel electrophoresis protocol with agarose native gel electrophoresis

A new protocol for conducting two-dimensional (2D) electrophoresis was developed by combining the recently developed agarose native gel electrophoresis with either vertical sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) or flat SDS agarose gel electrophoresis. Our innovative technique utilizes His/MES buffer (pH 6.1) during the first-dimensional (1D) agarose native gel electrophoresis, which allows for the simultaneous and clear visualization of basic and acidic proteins in their native states or complex structures. Our agarose gel electrophoresis is a true native electrophoresis, unlike blue native–PAGE, which relies on the intrinsic charged states of the proteins and their complexes without the need for dye binding. In the 2D, the gel strip from the 1D agarose gel electrophoresis is soaked in SDS and placed on top of the vertical SDS–PAGE gels or the edge of the flat SDS–MetaPhor high-resolution agarose gels. This allows for customized operation using a single electrophoresis device at a low cost. This technique has been successfully applied to analyze various proteins, including five model proteins (BSA, factor Xa, ovotransferrin, IgG, and lysozyme), monoclonal antibodies with slightly different isoelectric points, polyclonal antibodies, and antigen–antibody complexes, as well as complex proteins such as IgM pentamer and β-galactosidase tetramer. Our protocol can be completed within a day, taking approximately 5–6 h, and can be expanded further into Western blot analysis, mass spectrometry analysis, and other analytical methods.     

The simplified technique of high resolution two-dimensional polyacrylamide gel electrophoresis: biomedical applications in health and disease

The application of our simplified technique of high resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) to human body fluids is reviewed. Serum/plasma protein changes associated with alcohol abuse, familial dyslipoproteinemia ("fish-eye" disease), and myocardial infarction are demonstrated. High resolution 2-D PAGE of amniotic fluid, cerebrospinal fluid, urine, and saliva is shown with reference to the work of others, and the detection of pink-violet staining "lumicarmines" in sweat and tear fluid is reported for the first time. General aspects relating to the methodology are discussed. These include sample preparation, the choice of electrophoresis conditions (denaturing or nondenaturing) and detection method (Coomassie Brilliant Blue or silver), and the effects of native protein pretreatment with sodium dodecyl sulfate prior to silver staining or isoelectric focusing gel shrinkage in glycerol prior to second-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

A multichannel gel electrophoresis and continuous fraction collection apparatus for high‐throughput protein separation and characterization

To facilitate a direct interface between protein separation by PAGE and protein identification by mass spectrometry, we developed a multichannel system that continuously collects fractions as protein bands migrate off the bottom of gel electrophoresis columns. The device was constructed using several short linear gel columns, each of a different percent acrylamide, to achieve a separation power similar to that of a long gradient gel. A “Counter Free‐Flow” elution technique then allows continuous and simultaneous fraction collection from multiple channels at low cost. We demonstrate that rapid, high‐resolution separation of a complex protein mixture can be achieved on this system using SDS‐PAGE. In a 2.5 h electrophoresis run, for example, each sample was separated and eluted into 48–96 fractions over a mass range of ～10–150 kDa; sample recovery rates were 50% or higher; each channel was loaded with up to 0.3 mg of protein in 0.4 mL; and a purified band was eluted in two to three fractions (200 μL/fraction). Similar results were obtained when running native gel electrophoresis, but protein aggregation limited the loading capacity to about 50 μg per channel and reduced resolution.     

Identification of RFLP G6PD mutations by using microcapillary electrophoretic chips (Experion)

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most well-known human genetic defects, identified in more than 400 million individuals in the world. To date, no commercial kits are available for the mutation screening of this disease. Seventy G6PD-deficient Italian individuals admitted to the Laboratory of Clinical Molecular Biology of Hospital "Agostino Gemelli" of Rome were screened for the most frequent Italian mutations, by means of allele-specific PCR, followed by restriction fragment length electrophoresis. The present study compares two techniques for the identification of restriction patterns: agarose gel electrophoresis versus Experion system. When the first screening was negative, the entire G6PD gene was sequenced using the ABI 3100 Avant Instrumentation. The G6PD variants identified and their frequencies were the following: G6PD Mediterranean (75.7%), G6PD Seattle (7.1%), G6PD A(-) 202 + 376 (7.1%), and G6PD Cassano (2.8%). In addition, we identified by direct sequencing two new mutations, namely Buenos Aires and Rignano. With the Experion method, the size band determination was more accurate than that obtained by gel electrophoresis. The Experion system resulted as a valid, easy, and reproducible diagnostic method for the screening of G6PD mutation as compared with the agarose electrophoretic analysis.     

Ultrathin-layer sodium dodecyl sulfate gel electrophoresis of proteins: effects of gel composition and temperature on the separation of sodium dodecyl sulfate-protein complexes

This paper discusses the effects of gel composition and separation temperature on the migration properties of fluorescein-5-isothiocyanate-labeled protein molecular mass markers (ranging from 20 100 to 205 000 Da) in automated ultrathin-layer sodium dodecyl sulfate (SDS) gel electrophoresis. The separation mechanism with the agarose and composite agarose - linear polyacrylamide, agarose - hydroxyethyl cellulose, and agarose - polyethylene oxide matrices were all found to comply with the Ogston sieving model in the molecular mass range of the protein molecules investigated. Our temperature studies revealed that electrophoretic separation of SDS protein complexes is an activated process and, in pure agarose and in composite agarose hydroxyethyl cellulose and agarose - polyethylene oxide matrices that the separation requires increasing activation energy as a function of the molecular mass of the separated proteins. On the other hand, when linear polyacrylamide was used as composite additive, the activation energy demand of the separation decreased with increasing solute molecular mass. The sensitivity of the laser-induced fluorescent detection of the automated ultrathin-layer electrophoresis system was evaluated by injecting a series of dilutions of the markers and was found to be less than 2.5 ng/band for the fluorophore-labeled protein.     

Integration of isoelectric focusing with multi-channel gel electrophoresis by using microfluidic pseudo-valves

Two-dimensional (2D) protein separation is achieved in a plastic microfluidic device by integrating isoelectric focusing (IEF) with multi-channel polyacrylamide gel electrophoresis (PAGE). IEF (the first dimension) is carried out in a 15 mm-long channel while PAGE (the second dimension) is in 29 parallel channels of 65 mm length that are orthogonal to the IEF channel. An array of microfluidic pseudo-valves is created for introducing different separation media, without cross-contamination, in both dimensions; it also allows transfer of proteins from the first to the second dimension. Fabrication of pseudo-valves is achieved by photo-initiated, in situ gel polymerization; acrylamide and methylenebisacrylamide monomers are polymerized only in the PAGE channels whereas polymerization does not take place in the IEF channel where a mask is placed to block the UV light. IEF separation medium, carrier ampholytes, can then be introduced into the IEF channel. The presence of gel pseudo-valves does not affect the performance of IEF or PAGE when they are investigated separately. Detection in the device is achieved by using a laser induced fluorescence imaging system. Four fluorescently-labeled proteins with either similar pI values or close molecular weight are well separated, demonstrating the potential of the 2D electrophoresis device. The total separation time is less than 10 minutes for IEF and PAGE, an improvement of 2 orders of magnitude over the conventional 2D slab gel electrophoresis.     

Increased Resolution of Macromolecules with Agarose Gel Electrophoresis Compared with Polyacrylamide Gel Electrophoresis

Polyacrylamide gel electrophoresis (PAGE) is routinely used for electrophoretic characterization of biomolecules. In this study we demonstrate that this technique is quite useful for the characterization of small molecules, however, it becomes inadequate for the resolution of high‐molecular‐weight macromolecules like MUC2 mucins. We show that agarose gel electrophoresis (AGE) constitutes a better medium through which MUC2 macromolecules can penetrate and hence be resolved. MUC2 molecules obtained from PC/AA (a benign colonic adenoma cell line) were extracted, and their molecular weights (varying between 15–32 × 10⁶ Da) determined using Laser Light Scattering (LLS). These mucin molecules were reduced until 5–6 × 10⁶ subunits were obtained. The reduced and unreduced mucin molecules were run on an AGE and Western blotting and visualized, but were too large for PAGE. For this reason, the mucin subunits were treated with trypsin to obtain 500–600 kDa fragments. These tryptic fragments were then run on PAGE and AGE, and their resolutions compared. We conclude that AGE is a more useful electrophoretic method for characterization of macromolecules like mucin glycoproteins than PAGE.

Agarose gel electrophoresis of PC/AA cell layer extract (U: unreduced, intact form, R: reduced form).     

High-throughput genotyping of factor V Leiden mutation by ultrathin-layer agarose gel electrophoresis.

Ultrathin-layer agarose gel electrophoresis is a novel combination of the established methodologies of slab gel electrophoresis and capillary gel electrophoresis. This new format provides a multilane separation platform with rapid analysis time and excellent sensitivity by using laser-induced fluorescence scanning detection system. Sample injection onto the ultrathin-layer separation platform is easily accomplished by membrane mediated loading technology. In this paper, we demonstrate the sensitivity and high-throughput fashion of this novel separation and detection system for rapid genotyping of the coagulation factor V Leiden mutation by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis. The PCR amplified fragment from exon 10 of the factor V gene was digested by the Mnl I restriction enzyme, followed by automated ultrathin-layer agarose gel electrophoresis analysis with "in migratio" fluorescent labeling during the separation process. Due to its speed and automation, this method should be considered for large scale screening of factor V Leiden mutation.     

Novel moving reaction boundary-induced stacking and separation of human hemoglobins in slab polyacrylamide gel electrophoresis

We developed a novel polyacrylamide gel electrophoresis (PAGE) method to stack and separate human hemoglobins (Hbs) based on the concept of moving reaction boundary (MRB). This differs from the classic isotachophoresis (ITP)-based stacking PAGE in the aspect of buffer composition, including the electrode buffer (pH 8.62 Tris–Gly), sample buffer (pH 6.78 Tris–Gly), and separation buffer (pH 8.52 Tris–Gly). In the MRB-PAGE system, a transient MRB was formed between alkaline electrode buffer and acidic sample buffer, being designed to move toward the anode. Hbs carried partial positive charges in the sample buffer due to its pH below pI values of Hbs, resulting in electromigrating to the cathode. Hbs would carry negative charges quickly when migrated into the alkaline electrode buffer and be transported to the anode until meeting the sample buffer again. Thus, Hbs were stacked within a MRB until the transient MRB reached the separation buffer and then separated by zone electrophoresis with molecular sieve effect of the gel. The experimental results demonstrated that there were three clear and sharp protein zones of Hbs (HbA_1c, HbA₀, and HbA₂) in MRB-PAGE, in contrast to only one protein zone (HbA₀) in ITP-PAGE for large-volume loading (≥15 μl), indicating high stacking efficiency, separation resolution, and good sensitivity of MRB-PAGE. In addition, MRB-PAGE was performed in a conventional slab PAGE device, requiring no special device. Thus, it could be widely used in separation and analysis of diluted protein in a standard laboratory.

Improved resolution power of electrophoretic fractionation of DNA using a voltage gradient "up and down" application

The improved resolution power of electrophoretic fractionation of DNA in a wide range of molecular masses is demonstrated using an "up and down" application of voltage gradient gel electrophoresis (VGGE). This application also allows separation of different DNA fragments which are poorly fractionated in conventional electrophoresis.     

Combination of electrophoretic techniques for comprehensive analysis of complex protein systems

Analysis of proteins in complex mixtures such as cell lysates is presently performed by two-dimensional polyacrylamide gel electrophoresis under denaturing conditions (denaturing 2-D PAGE) followed by extraction of proteins from gel pieces and structural analysis of the proteins. This type of protein analysis is contributing to the correlation of information stored in DNA sequences with the structure of the product polypeptides. However, denaturing 2-D PAGE has its own limitations and it is necessary to develop various methods of protein analysis to reconstruct the total structure and function of proteins in complex systems. This review article summarizes the work in our laboratory to explore proteins in human plasma combining various electrophoretic techniques: nondenaturing and denaturing 2-D PAGE, capillary electrophoresis, and agarose gel isoelectric focusing.     

Miniaturization of Slab Gel Electrophoresis Using Thermal Lens Microscope and its Application to Genetic Diagnostics

Slab gel electrophoresis is the most widely used separation method for DNA fragments, proteins and carbohydrates, and miniaturization of this process is expected to provide fast, inexpensive and convenient analyses. However, two problems concerning the miniaturization of gel electrophoresis have to be solved:the separation performance and spatial resolution of the detector. We demonstrated that the separation performance was improved by using a discontinuous gel in which a concentrating gel was used to stack the sample to a sharp band, and using thermal lens microscope (TLM), which is highly sensitive and has a spatial resolution of micron level even in light scattering matrices as a gel, such sharpened separated bands were successfully detected. In this paper, we developed a miniaturized slab gel electrophoresis apparatus, demonstrated high speed separation of DNA fragments, and applied it to genetic diagnosis of coronary heart disease.     

Dodecyl maltoside detergent improves resolution of hepatic membrane proteins in two-dimensional gels.

This report describes the incorporation of an alkyl maltoside detergent in two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) sample lysis buffer in order to improve resolution of protein patterns separated by nonequilibrium pH gradient electrophoresis. Membrane-associated proteins with alkaline isoelectric points form horizontal streaks on two-dimensional electrophoretograms when solubilized with conventional nonionic detergent. Dodecyl maltoside enhances protein delipidation during solubilization and improves pattern resolution and protein mobility.     

A preliminary evaluation of serum proteins by capillary electrophoresis

We evaluated the 5-band Serum Proteins by Capillary Electrophoresis kit (Bio-Rad Laboratories, Hercules, CA, U.S.A.) on the BioFocus 2000 CE (Bio-Rad) against conventional agarose gel electrophoresis (AGE) (Helena Laboratories, Beaumont, TX, U.S.A.). Serum from 60 patients was initially screened by AGE and divided into three groups: 1) normal electrophoretic pattern (n = 36, mean total protein 67.7 g/L), 2) monoclonal/oligoclonal gammopathy (n = 14, mean total protein 78.8 g/L), and 3) polyclonal gammopathy (n = 10, mean total protein 77.4 g/L). These samples were concurrently analyzed on the BioFocus 2000. Intraassay and interassay CVs for the five fractions for a normal sample were 0.17-1.44% and 0.42-9.11%, respectively, and 0.21-3.37% and 0.29-3.61%, respectively, for a sample with monoclonal gammopathy. Correlation coefficients for albumin and the albumin/globulin (A/G) ratio were 0.8891 (albumin range 17-93 g/L) and 0.8276 (A/G ratio range 0.39-7.81), respectively. The A/G ratio alone could not discriminate between the three groups. Capillary zone electrophoresis (CZE) correctly identified 33 of 36 samples in the normal group, and 22 of 24 samples in the other two groups, giving a clinical sensitivity and specificity of 91.7%. Our preliminary evaluation shows that protein separation by CZE is a simple, rapid, and automated alternative to conventional AGE.     

Unlimited increase in the resolution of DNA ladders

Fractionation of DNA ladders by gel electrophoresis is limited by the progressive compressing of the long DNA end of a ladder. Improvement in the resolution of this DNA is achieved by use of the following two-step electrophoresis. Initially, the DNA ladder is fractionated by conventional constant field agarose gel electrophoresis. Subsequently, gel electrophoresis is performed in the reverse direction by pulsing the electrical field (PFGE). A newly developed type of pulsing is used, which causes inversion of a double-stranded DNA ladder: the distance migrated increases as the length of the DNA molecule increases. Thus, the resolution of DNA bands continues to increase during the PFGE. These two stages of electrophoresis are serially repeated. Eventually, both the short and the long DNA ends of the ladder migrate out of the gel while a selected region of the ladder undergoes progressive increase in resolution during back-and-forth migration. Improved resolution of DNA bands is achieved, without a known limit.     

Ultra-thin-layer agarose gel electrophoresis. I. Effect of the gel concentration and temperature on the separation of DNA fragments

A novel, rapid and efficient separation method is described for the analysis of double stranded (ds) DNA fragments in the form of horizontal ultra-thin-layer agarose gel electrophoresis. This separation technique combines the multilane, high-throughput separation format of agarose slab gel electrophoresis with the excellent performance of capillary electrophoresis. The electrophoretic separation of the fluorophore (Cy5)-labeled dsDNA molecules were imaged in real time by a scanning laser-induced fluorescence/avalanche photodiode detection system. Effects of the gel concentration (Ferguson plot) and separation temperature (Arrhenius plot) on the migration characteristics of the DNA fragments are discussed. An important genotyping application is also shown by characterizing the polymorphic region (2× or 4×48 base pair repeats) of the dopamine D₄ receptor gene (D₄DR, exon III region) for ten individuals, using PCR technology with Cy5-labeled primers and ultra-thin-layer agarose gel electrophoresis.     

High-throughput separation of DNA and proteins by three-dimensional geometry gel electrophoresis: feasibility studies

We report a novel separation method that is applicable to both DNA and protein samples, based on electrophoresis in a three-dimensional (3-D) geometry. In contrast to conventional electrophoresis, samples are applied in a two-dimensional, planar array to one of the surfaces of a 3-D geometry separation medium. Loading onto a plane results in a very high sample capacity. Sample migration and separation occur along the third spatial dimension, which is perpendicular to the loading plane. The key problem of electrophoresis in a 3-D geometry separation setup is that temperature gradients are caused by Joule's heat, affecting the electrical conductivity and viscosity of the separation medium. A means of achieving straight sample migration under these circumstances is to force heat flow through the separation medium parallel to the axis of sample migration. This can be done by dissipating the heat via the electrode sides of the gel and blocking any other heat transfer. The separation of DNA and proteins by this method has been tested using agarose gel electrophoresis, polyacrylamide gel electrophoresis, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Data were acquired off-line by conventional staining methods as well as on-line by detection of laser-induced fluorescence. We describe how to excise samples from the separation medium for preparative purposes. Possible unique applications of this 3-D geometry electrophoresis separation method are also discussed.     

High lane density slab-gel electrophoresis using micromachined instrumentation.

In this paper, micromachined pipette arrays (MPAs) and microcombs were studied as a means of enabling high lane density gel electrophoresis. The MPA provide a miniaturized format to interface sub-microliter volumes of samples between macroscale sample preparation formats and microscale biochemical analysis systems. The microcombs provide a means of creating sample loading wells in the gel material on the same center-to-center spacing as the MPAs. Together, the two micromachined instruments provide an alternative to current combs and pipetting technologies used for creating sample loading wells and sample delivery in gel electrophoresis systems. Using three designs for the microcomb-MPA pair, center-to-center spacings of 1.0 mm, 500 microm, and 250 microm are studied. The results demonstrate an approximate 10-fold increase in lane density and a 10-fold reduction in sample size from 5 microL to 500 pL. As a result, the number of theoretical plates has increased 2.5-fold, while system resolution has increased 1.5-fold over the conventional agarose gel systems. An examination of changes in resolution across the width of individual separation lanes in both systems revealed dependence in the case of the conventional gels and no dependence for the gels loaded with the micromachined instrumentation.     

Ionic liquid-assisted SDS-PAGE to improve human serum protein separation

Ionic liquid (IL)-assisted sodium dodecyl sulfate polyacrylamide gel electrophoresis (ILs-SDS-PAGE) was presented to improve protein separation. ILs were employed during the preparation process of polyacrylamide gel, then the modified gel was used for commercial protein marker, binary bovine serum albumin/lysozyme (BSA/Lyz) and human serum separation. The influence of ionic liquid concentration, cation alkyl chain length, cation and anion types on proteins separation were investigated. The results showed that ILs played a role in improving some protein separation, and ILs-SDS-PAGE provided higher resolution and separation efficiency than ordinary SDS-PAGE for low and middle relative molecular mass proteins in human serum. In addition, the principle of ILs-SDS-PAGE was discussed and the comparison of ILs-SDS-PAGE with ordinary SDS-PAGE and Native PAGE was made.