Cost‐effective media for the rapid and high resolution of small DNA fragments using polyacrylamide‐based electrophoresis

Current Tris‐based solutions for DNA electrophoresis produce a positive feedback loop between current and temperature at high voltage, resulting in long running times for the separation of even small DNA fragments. We optimized the separation of small DNA fragments (90–300 bp) in polyacrylamide‐based electrophoresis at high voltages (200volts/cm) by substituting Tris with low concentration alkali salts (e.g. 1 mm LiCl and CsCl). These media reduced the heat produced during electrophoresis, enhanced the DNA fragment resolution, and allowed gels to be run at higher voltages, reducing gel running times by 25%. In addition, the elimination of Tris and EDTA from the buffer reduced material costs approximately 10‐fold.     

Rapid agarose gel electrophoretic mobility shift assay for quantitating protein: RNA interactions

Interactions between proteins and nucleic acids are frequently analyzed using electrophoretic mobility shift assays (EMSAs). This technique separates bound protein:nucleic acid complexes from free nucleic acids by electrophoresis, most commonly using polyacrylamide gels. The current study utilizes recent advances in agarose gel electrophoresis technology to develop a new EMSA protocol that is simpler and faster than traditional polyacrylamide methods. Agarose gels are normally run at low voltages (∼10 V/cm) to minimize heating and gel artifacts. In this study we demonstrate that EMSAs performed using agarose gels can be run at high voltages (≥20 V/cm) with 0.5 × TB (Tris-borate) buffer, allowing for short run times while simultaneously yielding high band resolution. Several parameters affecting band and image quality were optimized for the procedure, including gel thickness, agarose percentage, and applied voltage. Association of the siRNA-binding protein p19 with its target RNA was investigated using the new system. The agarose gel and conventional polyacrylamide gel methods generated similar apparent binding constants in side-by-side experiments. A particular advantage of the new approach described here is that the short run times (5–10 min) reduce opportunities for dissociation of bound complexes, an important concern in non-equilibrium nucleic acid binding experiments.     

A simple and effective SuperBuffer for DNA agarose electrophoresis

In the paper, we describe a unique effective electrophoresis buffer for DNA agarose electrophoresis, called SuperBuffer. Using this buffer, electrophoresis could be performed within 10 min at voltages as high as 25 V/cm. In addition, DNA fragments of different lengths could be isolated clearly even at lower agarose gel concentrations and the DNA recovery efficiency was higher than that of the TAE/TBE running buffers. The SuperBuffer still retained its electrophoretic effect even after several uses.     

Separation of long RNA by agarose–formaldehyde gel electrophoresis

We describe a method to facilitate electrophoretic separation of high-molecular-weight RNA species, such as ribosomal RNAs and their precursors, on agarose–formaldehyde gels. Two alternative “pK-matched” buffer systems were substituted for the traditionally used Mops-based conductive medium. The key advantages include shortened run times, a 5-fold reduction in formaldehyde concentration, a significantly improved resolution of long RNAs, and consistency in separation. The new procedure has a streamlined work flow that helps to minimize errors and is broadly applicable to agarose gel electrophoresis of RNA samples and their subsequent analysis by Northern blotting.     

Rapid sequencing gel electrophoresis using glycerol-tolerant sodium taurine medium

We describe a new glycerol-tolerant sodium taurine (ST) medium for rapid sequencing gel electrophoresis by substituting the standard conductive media of Tris-boric acid-ethylenediaminetetraacetic acid (EDTA) (TBE) and Tris-taurine-EDTA (TTE) and other low-ionic-strength media of sodium boric acid (SB). Low-ionic-strength and cost-effective ST media gave glycerol tolerance up to 50% (v/v) glycerol-containing DNA sample solution, shorter running time, and better resolution to separate small DNA oligonucleotides (20-45 mer) in 12% denaturing sequencing gel electrophoresis.     

Ethidium DNA agarose gel electrophoresis: how it started

We started ethidium DNA agarose gel electrophoresis when our ultracentrifuge broke down and we needed an alternative method to check the quality of our mitochondrial DNA preparations. Agarose proved convenient for sizing DNA; ethidium in gel and buffer allowed visualization of DNA bands immediately after the run and improved the separation of the closed and open duplex forms of mitochondrial DNA circles. At smaller gel pore size mitochondrial DNA circles were excluded from the gel, whereas long linear DNAs were not. We concluded that the linear DNAs 'crawl like snakes head on through the gel'. This paper reviews some of the early experiments preceding the introduction of ethidium agarose gel electrophoresis.     

Analysis of supercoiled DNA by agarose gel electrophoresis using low-conducting sodium threonine medium

We describe a new low-ionic-strength sodium threonine (STh) medium with the advantage of avoiding relative DNA band migration changes following electrophoresis of supercoiled DNA in agarose gel when substituted for the standard conductive medium of TBE (Tris-boric acid-ethylenediaminetetraacetic acid [EDTA]) or TAE (Tris-acetic acid-EDTA) or the low-ionic-strength sodium boric acid medium. Low-ionic-strength STh medium provided better resolution, less heat generation, and prevention of relative migration order changes among linear, covalently closed circular-, and open circular-formed DNA in the range of 2-10 kilobase pairs in 1% agarose gel electrophoresis.     

Effect of beta-cyclodextrin on the renaturation of enzymes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Activity gel assays require a long incubation time (several hours) on renaturation of enzymatic activity after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). To reduce the incubation time, we used a novel renaturation buffer containing cyclic oligosaccharide β-cyclodextrin (β-CD) which is capable of capturing SDS. Yeast α-glucosidase, used as a model protein, was run on SDS-PAGE, and then the gel matrix was incubated in a variety of renaturation buffers. Compared with conventional renaturation buffers containing Triton X-100 or isopropanol, our novel renaturation buffer containing β-CD can restore enzymatic activity within 10 min. Therefore, this new format represents a good alternative with reduced incubation time for activity gel assays.     

Separation of very large DNA molecules by gel electrophoresis.

Very large DNA molecules were separated by electrophoresis in horizontal slab gels of dilute agarose. Conditions of electrophoresis were developed using intact DNA molecules from the bacterial viruses lambda, T4 and G. Their DNAs have molecular weights (M) of 32 million, 120 million, and 500 million, respectively. Several electrophoresis conditions were found which give sufficiently high mobilities and large differences that these DNAs are separated in a short time. Electrophoresis in 0.1% agarose at 2.5 V/cm of gel length separates T4 and lambda DNAs by 2.0 cm, and G and T4 DNAs by 1.0 cm in only 10 hr. With some conditions DNA mobilities are directly proportional to log M for M values from 10 to 500 million. The procedures used will allow rapid molecular weight determination and separation of very large DNA molecules.     

Plant protein isolation and stabilization for enhanced resolution of two-dimensional polyacrylamide gel electrophoresis

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is the common method of choice for proteomic analysis. By introducing several small changes, a method was developed that not only improved the resolution and reproducibility of 2D-PAGE but also shortened the time of analysis. Precipitation by alkaline phenol and methanol/ammonium acetate was the choice for protein extraction. However, instead of precipitating the proteins overnight at -20 °C, it was carried out for 2 to 3 h at -80 °C. Ethanol was used for the final wash of the protein precipitate instead of routinely used acetone. Dithiothreitol (DTT) was used in all solutions from the beginning, considerably improving the solubilization of precipitated proteins. Solubilization was further improved by using a mixture of detergents and denaturants at high concentrations along with large amounts of DTT. Both in-gel rehydration and cup-loading methods were used for isoelectric focusing (IEF). For in-gel rehydration, samples reduced with DTT were diluted with sample buffer containing 2-hydroxyethyl disulfide (2-HED) (1:3) or were cup-loaded on a strip rehydrated with sample buffer containing 2-HED. Glycerol (5%) was used in the sample buffer, and the focusing was performed at 15 °C. The applicability of the method was demonstrated using several soybean tissues.     

Improved resolution with one-dimensional polyacrylamide gel electrophoresis: myofibrillar proteins from typed single fibers of human muscle

Standard procedures for one-dimensional discontinuous sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and silver staining were modified to give more effective separation and an improved resolution of human skeletal muscle proteins. In this system, an electrophoresis buffer composed of 100 mM L-isoleucine, 25 mM Tris base, and 0.1% SDS was used. The separating gel consisted of 16% acrylamide with N,N'-methylenebisacrylamide as a crosslinker (1:23), 0.4% SDS, 1.5 M Tris-HCl, pH 8.8. By the present procedure, the slow and the fast forms of myosin light chains (LCs, LCf) and other contractile proteins from human muscle could be better separated. The silver stain is based on a combination of methods previously described. The modified method requires a small fragment of a single fiber to observe as few as 10 ng of myofibrillar muscle proteins. The described simplifications made it possible to assay and compare up to 40 single fibers in the same electrophoretic run. Improved separation of other proteins migrating at basic pH could be achieved by a similar approach.     

产热凝胶土壤杆菌ATCC31749蛋白质组双向电泳图谱的构建

建立了热凝胶生产茵土壤杆茵茵体总蛋白的蛋白质提取方法和双向电泳方案,确定了使用蛋白质裂解液(7 mol/L尿素,2 mol/L硫脲,1%ASB-14去垢剂,40 mmol/L Tris,0.001%溴酚蓝,1 mmol/L EDTA,1%TBP和1%两性电解质)结合超声破碎法来提取茵体总蛋白的方案为最佳,选择17 cm...     

Investigation of the repair of single-strand breaks in human DNA using alkaline gel electrophoresis

Unstimulated lymphocytes from eight healthy persons were exposed to 10-, 30-, and 100-Gy doses of 60Co gamma radiation. The repair of damaged DNA was measured by (1) alkaline gel electrophoresis (extracted DNA loaded on 0.25% agarose gel, run at 1 V/cm for 39-44 h) at 0, 1, and 2 h after exposure and (2) incorporation of [3H]thymidine into unstimulated lymphocytes in the presence of 2 mM hydroxyurea 1 and 2 h after exposure. Both methods--alkaline gel electrophoresis and thymidine incorporation--showed that repair was completed within 2 h.     

Electrophoresis time impacts the denaturing gradient gel electrophoresis-based assessment of bacterial community structure

We investigated the impact of denaturing gradient gel electrophoresis (DGGE) run time on the assessment of bacterial community structure. Results indicated that increased electrophoresis run time (while maintaining 1000 volt-hours) resulted in dissimilar profiles, likely due to instability of the denaturing gradient. We recommend that DGGE run times be minimized to provide optimal band resolution, as extended electrophoresis times can greatly impact subsequent band-based analyses.     

Purification of a non-chloroplastic α-glucan phosphorylase from spinach leaves

The non-chloroplastic -glucan phosphorylase (EC 2.4.1.1) from spinach leaves has been purified to homogeneity as revealed by dodecylsulfate gel electrophoresis. Both purification and separation from the chloroplastic phosphorylase were achieved by chromatography on Sepharose-bound dextrin. The chloroplastic phosphorylase did not bind to Sepharose-dextrin and was removed from the column by washing with buffer, as verified by polyacrylamide gel electrophoresis of the buffer eluate and by chromatography of a preparation from isolated intact chloroplasts. The non-chloroplastic phosphorylase did bind to a high extent to Sepharose-dextrin and could be eluted by a dextrin gradient. Based on dodecylsulfate gel electrophoresis and pyridoxal phosphate determination, a molecular weight of about 90,000 was found for the monomer. Molecular-weight determination by porosity density gradient electrophoresis and gel filtration on Sephadex G-200 suggested that the native enzyme is a dimer, as are other phosphorylases.Abbreviations DEAE diethylaminoethyl - EDTA ethylenediamine tetraacetic acid - G1P glucose 1-phosphate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - PMSF phenylmethyl sulphonyl fluoride - SDS sodium dodecylsulfate - Tris Tris (hydroxymethyl)aminomethane Dedicated to Professor Dr. A. Pirson on the occasion of his 70th birthday     

Single universal primer multiplex ligation-dependent probe amplification with sequencing gel electrophoresis analysis

In this study, a novel single universal primer multiplex ligation-dependent probe amplification (SUP-MLPA) technique that uses only one universal primer to perform multiplex polymerase chain reaction (PCR) was developed. Two reversely complementary common sequences were designed on the 5′ or 3′ end of the ligation probes (LPs), which allowed the ligation products to be amplified through only a single universal primer (SUP). SUP-MLPA products were analyzed on sequencing gel electrophoresis with extraordinary resolution. This method avoided the high expenses associated with capillary electrophoresis, which was the commonly used detection instrument. In comparison with conventional multiplex PCR, which suffers from low sensitivity, nonspecificity, and amplification disparity, SUP-MLPA had higher specificity and sensitivity and a low detection limit of 0.1 ng for detecting single crop species when screening the presence of genetically modified crops. We also studied the effect of different lengths of stuffer sequences on the probes for the first time. Through comparing the results of quantitative PCR, the LPs with different stuffer sequences did not affect the ligation efficiency, which further increased the multiplicity of this assay. The improved SUP–MLPA and sequencing gel electrophoresis method will be useful for food and animal feed identification, bacterial detection, and verification of genetic modification status of crops.     

Capillary gel electrophoresis for rapid, high resolution DNA sequencing.

Capillary gel electrophoresis has been demonstrated for the separation and detection of DNA sequencing samples. Enzymatic dideoxy nucleotide chain termination was employed, using fluorescently tagged oligonucleotide primers and laser based on-column detection (limit of detection is 6,000 molecules per peak). Capillary gel separations were shown to be three times faster, with better resolution (2.4 x), and higher separation efficiency (5.4 x) than a conventional automated slab gel DNA sequencing instrument. Agreement of measured values for velocity, resolution and separation efficiency with theory, predicts further improvements will result from increased electric field strengths (higher voltages and shorter capillaries). Advantages of capillary gel electrophoresis for automatic DNA sequencing instruments and for genomic sequencing are discussed.     

Process optimisation for anion exchange monolithic chromatography of 4.2 kbp plasmid vaccine (pcDNA3F)

Anion exchange monolithic chromatography is increasingly becoming a prominent tool for plasmid DNA purification but no generic protocol is available to purify all types of plasmid DNA. In this work, we established a simple framework and used it to specifically purify a plasmid DNA model from a clarified alkaline-lysed plasmid-containing cell lysate. The framework involved optimising ligand functionalisation temperature (30–80 °C), mobile phase flow rate (0.1–1.8 mL/min), monolith pore size (done by changing the porogen content in the polymerisation reaction by 50–80%), buffer pH (6–10), ionic strength of binding buffer (0.3–0.7 M) and buffer gradient elution slope (1–10% buffer B/min). We concluded that preferential pcDNA3F adsorption and optimum resolution could be achieved within the tested conditions by loading the clarified cell lysate into 400 nm pore size of monolith in 0.7 M NaCl (pH 6) of binding buffer followed by increasing the NaCl concentration to 1.0 M at 3%B/min.     

Recovery of active N-acetyl-D-glucosamine 2-epimerase from inclusion bodies by solubilization with non-denaturing buffers

Overexpression of recombinant N-acetyl-d-glucosamine 2-epimerase, one of the key enzymes for the synthesis of N-acetylneuraminic acid, in E. coli led to the formation of protein inclusion bodies. In this study we report the recovery of active epimerase from inclusion bodies by direct solubilization with Tris buffer. At pH 7.0, 25% of the inclusion bodies were solubilized with Tris buffer. The specific activity of the solubilized proteins, 2.08 ± 0.02 U/mg, was similar to that of the native protein, 2.13 ± 0.01 U/mg. The result of circular dichroism spectroscopy analysis indicated that the structure of the solubilized epimerase obtained with pH 7.0 Tris buffer was similar to that of the native epimerase purified from the clarified cell lysate. As expected, the extent of deviation in CD spectra increased with buffer pH. The total enzyme activity recovered by solubilization from inclusion bodies, 170.41 ± 10.06 U/l, was more than 2.5 times higher than that from the clarified cell lysate, 67.32 ± 5.53 U/l. The results reported in this study confirm the hypothesis that the aggregation of proteins into inclusion bodies is reversible and suggest that direct solubilization with non-denaturing buffers is a promising approach for the recovery of active proteins from inclusion bodies, especially for aggregation-prone multisubunit proteins.     

Transferrin variants in sheep: separation and characterization by polyacrylamide gel electrophoresis and isoelectric focusing

Isoelectric focusing with carrier ampholytes in ultrathin polyacrylamide gels and polyacrylamide gel electrophoresis in a discontinuous buffer system were used for the separation of sheep transferrin variants. For identification of the different iron-binding sites of transferrin a stepwise urea gradient, different degrees of iron saturation and double one-dimensional electrophoresis were used. Isoelectric focusing results in an increased resolution of the Fe0-transferrin, Fe1-transferrin and Fe2-transferrin region. At the level of Fe0-transferrin and Fe1-transferrin the variants I, A, G, B, C, D, M, E, Q, P can be identified. The method is especially suitable for genetic studies. For screening purposes up to 108 samples can be separated within one run in an ultrathin gel.