Production of polyacrylamide gel filled capillaries for capillary gel electrophoresis (CGE): Influence of capillary surface pretreatment on performance and stability

Polyacrylamide gel filled capillaries for electrophoresis can be improved in performance and stability by a specially optimized and reproducible production procedure. After surface pretreatment as described in this contribution, gels polymerized in capillaries are stable for long series of routine separations. Bubbles formation during gel polymerization can be avoided. The procedure is compared with those described by other authors. Separations of different oligonucleotide samples illustrate the quality of the gel capillaries obtained. The peak symmetry achieved proved to be better than that obtained without surface pretreatment.     

Vertical agarose gel electrophoresis and electroblotting of high-molecular-weight proteins

The electrophoretic separation of high-molecular-weight proteins (> 500 kDa) using polyacrylamide is difficult because gels with a large enough pore size for adequate protein mobility are mechanically unstable. A 1% vertical sodium dodecyl sulfate (SDS)-agarose gel electrophoresis (VAGE) system has been developed that allows titin (a protein with the largest known SDS subunit size of 3000-4000 kDa) to migrate over 10 cm in a approximately 13 cm resolving gel. Such migration gives clear and reproducible separation of titin isoforms. Proteins ranging in size from myosin heavy chain ( approximately 220 kDa) up to titin can be resolved on this gel system. Electroblotting of these very large proteins was nearly 100% efficient. This VAGE system has revealed two titin size variants in rabbit psoas muscle, two N2BA bands in rabbit cardiac muscle, and species differences between titins from rat and rabbit muscle. Agarose electrophoresis should be the method of choice for separation and blotting of proteins with very large subunit sizes.     

Direct and specific recognition of glycosaminoglycans by antibodies after their separation by agarose gel electrophoresis and blotting on cetylpyridinium chloride-treated nitrocellulose membranes

A method for the immunodetection of several natural complex polysaccharides (glycosaminoglycans) after their separation by conventional agarose gel electrophoresis, blotting and immobilizing on nitrocellulose membranes derivatized with the cationic detergent cetylpyridinium chloride (CPC), and direct and specific immunodetection by antibodies is described. This new approach is based on the principles that were used to develop the Western blot, and is applied to the separation of the glycosaminoglycans purified from normal human urine. After migration in agarose gel electrophoresis, chondroitin sulfate samples of different origin were blotted and transferred onto nitrocellulose membranes treated with CPC. Immunodetection was performed using the anti-chondroitin-6-sulfate antibody that specifically recognizes intact chondroitin-6-sulfate. By calculating the ratio between the antibody staining (epitope) and alcian blue staining (mass), the epitope density expressed as a percentage, i.e., the number of repetitive epitopes per mass, was obtained. These values were in agreement with the quantitation of 6-sulfated groups of chondroitin sulfate performed by the evaluation of unsatured disaccharide-6-sulfate (DeltaDi6S) produced after treatment with chondroitinase ABC and separated by high-performance liquid chromatography (HPLC). Furthermore, immunodetection of heparan sulfate was performed using the anti-heparan sulfate antibody.     

Polyacrylamide gel electrophoresis of fluorophore-labeled hyaluronan and chondroitin sulfate disaccharides: application to the analysis in cells and tissues

This report describes a new formulation of polyacrylamide gel electrophoresis of fluorophore-labeled saccharides (PAGEFS) for the analysis of hyaluronan (HA) and chondroitin sulfate (CS) Delta-disaccharides. PAGEFS relies on derivatization of reducing ends of HA- and the variously sulfated CS-derived Delta-disaccharides with 2-aminoacridone (AMAC), followed by electrophoresis under optimized buffer conditions (Tris-borate and Tris-HCl) and on polyacrylamide gels (25% T/3.75% C). The method was applied to the analysis of glycosaminoglycans (GAGs) from the human umbilical cord tissue and GAGs isolated from human aortic smooth muscle cell cultures. The obtained results were in agreement with those obtained after an analysis with high-performance liquid chromatography (HPLC). On the basis of these results, PAGEFS is a rapid and sensitive method for the analysis of the total amount of HA- and CS-derived disaccharides, as it allows analyzing 20 samples in minigels in one run and provides quantitation with relatively high sensitivity (less than 25 pmol per disaccharide). In addition, PAGEFS overcomes the lack of commercial gels described previously for the separation of AMAC-labeled disaccharides. Therefore, the method proposed here is an economic and useful tool for a fast screening of GAGs in biological samples, particularly when a high number of samples should be analyzed.     

Detection of submicrogram quantities of glycosaminoglycans on agarose gels by sequential staining with toluidine blue and Stains-All

A sensitive method has been developed for the visualization of nonradiolabelled glycosaminoglycans resolved by agarose gel electrophoresis using staining with toluidine blue followed by Stains-All procedure. This method, which can detect as little as 10 ng of a single species, can be used to stain a few micrograms of a complex polysaccharide mixture. The combination of agarose gel electrophoresis and sequential toluidine blue/Stains-All staining can be applied to the analysis of all the complex glycosaminoglycans (i.e., heparin, heparan sulfate, chondroitin/dermatan sulfate) and nonsulfated polyanions (i.e., hyaluronate, defructosylated capsular polysaccharide K4) as well as to comparisons of specificities of the glycosaminoglycan-degrading enzymes and the identification and quantification of the contaminations of other polysaccharides within glycosaminoglycan preparations with great sensitivity (about 0.1%). Furthermore, this method can be used to stain low-molecular-mass fractions and oligosaccharides derived from the natural polyanions, such as heparin. This procedure may be particularly valuable in situations where the availability of glycosaminoglycan is very limited.     

On-line coupling of gel electrophoresis and inductively coupled plasma-mass spectrometry

The on-line coupling of gel electrophoresis with inductively coupled plasma-mass spectrometry (GE-ICP-MS) is a powerful tool for simultaneous separation, detection and quantification of bio-molecules, and has been applied to the determination of phosphorus in DNA, phosphoproteins, and phosphopeptides, gold in nano-particles, iron in metalloproteins, and iodine in aerosols, and cisplatin-oligonucleotide interactions. However, since the first report in 2005, relatively few papers have been published, perhaps reflecting the lack of familiarity with the benefits of this promising methodology. So, here for the first time, we critically review the applications of GE-ICP-MS, and explore the advantages and the limitations of the technique for various applications. Such scrutiny may be useful in not only the development of the technique but also highlighting its potential in proteomics, genomics and metallomics.     

High-sensitive analysis of DNA fragments by capillary gel electrophoresis using transient isotachophoresis preconcentration and fluorescence detection

In this report aimed on further development of a high-sensitivity capillary gel electrophoresis (CGE) method for analysis of DNA fragments, we firstly explored online transient isotachophoresis (tITP) preconcentration combined with fluorescence detection (FD). The fluorescence signal (excitation: 488 nm; emission: 590 nm) was generated using the intercalating dye of ethidium bromide (EB). It was found when the leading electrolyte (LE) was injected behind the sample zone, such a special tITP mode has significant advantages to solve the bubble formation issue and to improve the analytical performance stability. Two standard DNA samples, a 50 bp DNA step ladder and the φX174/HaeIII digest, were used to evaluate the qualitative and quantitative abilities of the tITP-FD approach. A highly diluted sample (10,000-fold in the water, e.g. the φX174/HaeIII digest diluted from 500 μg/ml to the 50 ng/ml level) was enriched and detected; the LOD was down to 0.09 ng/ml for the 72 bp fragment, apparently improved more than 1000-fold in comparison with UV detection. Although the RSD of peak areas (n = 3) was around 15.5% for the sample was electrokinetically injected, good linearity of peak area response showed that the proposed method is suitable for quantitative analysis.     

Evaluation of agarose gel electrophoresis for characterization of silver nanoparticles in industrial products

Agarose gel electrophoresis (AGE) has been used extensively for characterization of pure nanomaterials or mixtures of pure nanomaterials. We have evaluated the use of AGE for characterization of Ag nanoparticles (NPs) in an industrial product (described as strong antiseptic). Influence of different stabilizing agents (PEG, SDS, and sodium dodecylbenzenesulfonate), buffers (TBE and Tris Glycine), and functionalizing agents (mercaptosuccinic acid (TMA) and proteins) has been investigated for the characterization of AgNPs in the industrial product using different sizes‐AgNPs standards. The use of 1% SDS, 0.1% TMA, and Tris Glycine in gel, electrophoresis buffer and loading buffer led to the different sizes‐AgNPs standards moved according to their size/charge ratio (obtaining a linear relationship between apparent mobility and mean diameter). After using SDS and TMA, the behavior of the AgNPs in the industrial product (containing a casein matrix) was completely different, being not possible their size characterization. However we demonstrated that AGE with LA‐ICP‐MS detection is an alternative method to confirm the protein corona formation between the industrial product and two proteins (BSA and transferrin) maintaining NPs‐protein binding (what is not possible using SDS‐PAGE).     

Inverse-flow derivatization for capillary electrophoresis of DNA fragments with laser-induced fluorescence detection

A novel method is presented to detect DNA fragments separated by capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection using inverse-flow derivatization. In electrophoresis, the intercalating dye, thiazol orange was only added to the separation buffer at the positive polarity. The negatively charged DNA fragments migrated from the negative polarity to the positive polarity, while the positively charged dye migrated in the opposite direction. When DNA fragments met with dye ions, the DNA–dye complexes were formed. The complexes continued migrating to the positive end, due to their net negative charges. When the complexes passed through the detection window, the fluorescent signals were generated. Importantly, DNA fragments migrated as their native state before DNA–dye complexes were formed. This procedure was used to detect double stranded DNA (dsDNA) and single stranded DNA (ssDNA) fragments, and polymerase chain reaction (PCR) products. The excellent resolution and good reproducibility of DNA fragments were achieved in non-gel sieving medium. This procedure may be useful in genetic mutation/polymorphism detections.     

Quantitative-competitive polymerase chain reaction coupled with slab gel and capillary electrophoresis for the detection of roundup ready soybean and maize

The aim of the present study was to develop a quantitative-competitive PCR (QC-PCR) method to detect DNA from transgenic herbicide-resistant (roundup ready, RR) soybean and maize. Since no QC-PCR system for the quantification of RR maize had been published at the time of writing, a specific competitor DNA for transgenic event was developed. For the QC-PCR of RR-soybean, a commercially available competitor was employed. These internal standards were calibrated by coamplifying with mixtures containing RR-soybean and maize DNAs. The calibrated QC-PCR systems were applied to certified RR-soybean and maize flour mixtures in order to demonstrate their suitability not only for the quantification of the glyphosate resistance traits in DNA matrices, but also in practically relevant samples. In addition, a special focus of the present work was to compare the detection of QC-PCR products by slab gel and CGE with UV detection. CGE permitted the precise detection of transgenic events also below the equivalence points; while in slab gel electrophoresis, due to the low sensitivity the quantification of genetically modified DNA was allowed only at the equivalence point.     

毛细管电泳聚乙烯吡咯烷酮与羟乙基纤维素混配无胶筛分介质分离DNA片段

本文报道了毛细管电泳聚乙烯吡咯烷酮与羟乙基纤维素混配无胶筛分介质分离较短的 p GEM7Zf(+) Hae DNA片段 (DNA长度为 1 8～ 675bp)。研究表明 ,在 1 %的羟乙基纤维素无胶筛分介质中 ,加入 2 %的聚乙烯吡咯烷酮能显著提高 DNA片段的分辨率和分离效率。在混配无胶筛分介质中 ,聚乙烯吡咯烷酮有两种作用 ,一是动态涂渍 ,降低毛细管内壁对 DNA片段与 DNA荧光插入试剂的吸附 ,改善分离效率 ;二是两种不同长度、性质的线性高分子能形成更为致密的“缠绕网络”,有利于较短的 DNA片段电泳分离。     

Quantitative analysis of DNA methylation in the promoter region of the methylguanine‐O6‐DNA‐methyltransferase gene by COBRA and subsequent native capillary gel electrophoresis

Along with histone modifications, RNA interference and delayed replication timing, DNA methylation belongs to the key processes in epigenetic regulation of gene expression. Therefore, reliable information about the methylation level of particular DNA fragments is of major interest. Herein the methylation level at two positions of the promoter region of the gene methylguanine‐O⁶‐DNA‐Methyltransferase (MGMT) was investigated. Previously, it was demonstrated that the epigenetic status of this DNA region correlates with response to alkylating anticancer agents. An automated CGE method with LIF detection was established to separate the six DNA fragments resulting from combined bisulfite restriction analysis of the methylated and non‐methylated MGMT promoter. In COBRA, the DNA was treated with bisulfite converting cytosine into uracil. During PCR uracil pairs with adenine, which changes the original recognition site of the restriction enzyme Taql. Artificial probes generated by mixing appropriate amounts of DNA after bisulfite treatment and PCR amplification were used for validation of the method. The methylation levels of these samples could be determined with high accuracy and precision. DNA samples prepared by mixing the corresponding clones first and then performing PCR amplification led to non‐linear correlation between the corrected peak areas and the methylation levels. This effect is explained by slightly different PCR amplification of DNA with different sequences present in the mixture. The superiority of CGE over PAGE was clearly demonstrated. Finally, the established method was used to analyze the methylation levels of human brain tumor tissue samples.     

Development and application of a capillary electrophoresis based method for the simultaneous screening of six antibiotics in spiked milk samples

An easy to use and low time consuming capillary electrophoresis (CE) method was developed and applied to the simultaneous determination of six antibiotics (ampicillin, amoxicillin, cloxacillin, penicillin, tetracycline and chloramphenicol) in spiked milk samples. Samples of milk were cleaned up by solid-phase extraction (with a C₁₈ cartridge) after protein precipitation. Analysis was performed by CE and results compared with the obtained via HPLC, both coupled to a UV-vis detector (210 nm). CE employed a 58.5 cm long fused-silica capillary (50 cm to detector), 75 μm i.d., a 2.7 × 10⁻² M KH₂PO₄, 4.3 × 10⁻² M Na₂B₄O₇ separation buffer, pH 8; an applied voltage of 18 kV; a hydrostatic injection of 0.5 psi during 3 s; and a run temperature of 25 °C. Under the described conditions, amoxicillin was not separated by HPLC, while CE was able to separate, and, therefore, allow detection. Regardless of amoxicillin, comparable results were obtained by HPLC and CE. The average recoveries of antibiotics, from milk fortified at 2.5 and 5 μg/mL, was over 72% with R.S.D.s within 5%. Recovery levels were essentially dictated by the used SPE cartridge.     

Nanoparticle-filled capillary electrophoresis for the separation of long DNA molecules in the presence of hydrodynamic and electrokinetic forces

We report the analysis of long DNA molecules by nanoparticle-filled capillary electrophoresis (NFCE) under the influences of hydrodynamic and electrokinetic forces. The gold nanoparticle (GNP)/polymer composites (GNPPs) prepared from GNPs and poly(ethylene oxide) were filled in a capillary to act as separation matrices for DNA separation. The separations of lambda-DNA (0.12-23.1 kbp) and high-molecular-weight DNA markers (8.27-48.5 kbp) by NFCE, under an electric field of -140 V/cm and a hydrodynamic flow velocity of 554 microm/s, were accomplished within 5 min. To further investigate the separation mechanism, the migration of lambda-DNA was monitored in real time using a charge-coupled device (CCD) imaging system. The GNPPs provide greater retardation than do conventional polymer media when they are encountered during the electrophoretic process. The presence of interactions between the GNPPs and the DNA molecules is further supported by the fluorescence quenching of prelabeled lambda-DNA, which occurs through an energy transfer mechanism. Based on the results presented in this study, we suggest that the electric field, hydrodynamic flow, and GNPP concentration are the three main determinants of DNA separation in NFCE.     

On-line sample enrichment for the determination of proteins by capillary zone electrophoresis with poly(vinyl alcohol)-coated bubble cell capillaries

Field-amplified sample stacking (FASS) is used to separate basic proteins in a poly-(vinyl alcohol)-coated bubble cell capillary. To our knowledge, this is the first paper describing the on-column stacking of proteins (as cations) using FASS in bubble cell capillary. The bubble cell capillary is fabricated using a one-step method. Cetyltrimethylammonium chloride is added into the running buffer to reverse the EOF and, thus, to pump the water plug out during the sample stacking step. The effect of the water plug lengths and sample injection durations were investigated and optimized. The results obtained were compared with those for the normal capillary without bubble cell in terms of resolution and sensitivity enhancement. Under the optimal condition, this method can improve the sensitivity of the peak areas ranging from 5000- to 26 000-fold. The RSDs (n = 5) of the migration time and peak area are satisfactory (less than 0.6 and 12%, respectively). Application of the capillary electrophoresis method with bubble cell, FASS, and UV detection thereby leads to the determination of these proteins at concentrations ranging from 3 to 10 ng/mL, based on a signal-to-noise ratio of 3:1.     

Characterization of AMA1-RON2L complex with native gel electrophoresis and capillary isoelectric focusing

Rhoptry neck protein 2 (RON2) binds to the hydrophobic groove of apical membrane antigen 1 (AMA1), an interaction essential for invasion of red blood cells (RBCs) by Plasmodium falciparum (Pf) parasites. Vaccination with AMA1 alone has been shown to be immunogenic, but unprotective even against homologous challenge in human trials. However, the AMA1-RON2L (L is referred to as the loop region of RON2 peptide) complex is a promising candidate, as preclinical studies with Freund's adjuvant have indicated complete protection against lethal challenge in mice and superior protection against virulent infection in Aotus monkeys. To prepare for clinical trials of the AMA1-RON2L complex, identity and integrity of the candidate vaccine must be assessed, and characterization methods must be carefully designed to not dissociate the delicate complex during evaluation. In this study, we developed a native Tris-glycine gel method to separate and identify the AMA1-RON2L complex, which was further identified and confirmed by Western blotting using anti-AMA1 monoclonal antibodies (mAbs 4G2 and 2C2) and anti-RON2L polyclonal Ab coupled with mass spectrometry. The formation of complex was also confirmed by Capillary Isoelectric Focusing (cIEF). A short-term (48 h and 72 h at 4°C) stability study of AMA1-RON2L complex was also performed. The results indicate that the complex was stable for 72 h at 4°C. Our research demonstrates that the native Tris-glycine gel separation/Western blotting coupled with mass spectrometry and cIEF can fully characterize the identity and integrity of the AMA1–RON2L complex and provide useful quality control data for the subsequent clinical trials.     

Microchip capillary electrophoresis with electrochemical detector for precolumn enzymatic analysis of glucose, creatinine, uric acid and ascorbic acid in urine and serum

An integrated multiple-enzymatic assay was performed on a (microchip capillary electrophoresis) μCE-EC chip capable of precise intake of sample or reagents in nanoliters. Incorporating multiple-enzyme assay into the μCE chip is relatively new—rendering simultaneous analysis of creatinine and uric acid a snap.Added to the list of merits in this study are the enhanced sensitivity down to 1 μM and a broader spectrum of analytes—inclusive of glucose for the long-time sufferers of diabetes. The performance was orchestrated to attain the claimed level: employing the end-channel electrode mode to tame the noises and the precolumn enzymatic reaction to stabilize the baseline. The 10 μm embedded Pt electrode, deposited at the end of the 30 μm wide separation channel, benefited chip fabrication besides noise reduction. The optimized conditions were 20 mM phosphate buffer (pH 7.5), +1.5 kV separation voltage and +1.0 V detection potential (versus Ag/AgCl). The migration time was repeatable within the deviation of 0.5% R.S.D. (n=7), but the peak currents ranged from 1.5 to 2.2% R.S.D. The detection limits (S/N=3) ranged from 0.71 μM for ascorbic acid to 10 μM for glucose. The calibration curve was linear from 10 to 800 μM (R²>0.995). Glucose, creatinine, uric acid and ascorbic acid as model analytes, in pure form or in serum and urine samples, were tested to verify its feasibility.