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.     

Size separation of proteins by capillary zone electrophoresis with cationic hitchhiking

The paper describes a method of size separation of proteins by capillary sieving electrophoresis with cationic surfactant. Proteins are separated within 12 min with repeatability of migration times better than 0.2%. Some proteins achieve the separation efficiency of 200,000 theoretical plates. The method can be used for determination of protein relative molecular masses. The accuracy of the determined relative molecular masses and the limitation of the method were investigated by the analysis of more than 60 proteins. The method also allows separation of protein oligomers. Proteins can be quantitated after the electrokinetic injection in the concentration range 0.07-0.43?g/L. The average detection limit is about 2?mg/L.     

Two-dimensional gel electrophoresis/matrix-assisted laser desorption/ionisation mass spectrometry of commercial bovine milk

Proteins in commercial bovine milk have been separated by two-dimensional gel electrophoresis and examined by matrix-assisted laser desorption/ionisation mass spectrometry. Gel separation was conducted in two different pH gradients, 3-10 and 6-11; the latter range resulted in a higher spot resolution and favoured the basic proteins. We have limited the time-of-flight mass spectrometry analysis to the linear mode to examine the capability of reliable relative molecular masses of the intact proteins in their characterisation. The present study draws attention to the difficulty of identifying basic proteins with low molecular masses (below 12000 Da) that are commonly encountered in milk samples.     

Preparative high-yield electroelution of proteins after separation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and its application to analysis of amino acid sequences and to raise antibodies.

A method for the preparative high-yield electroelution of proteins from sodium dodecyl sulphate (SDS) polyacrylamide gel strips was established. The method consisted of SDS-polyacrylamide gel electrophoresis, detection of proteins with sodium acetate and electrophoretic elution at 200 V for 3 h by utilizing a horizontal flat-bed gel electrophoresis apparatus. Standard proteins with molecular masses of 14-66 kilodalton (cytochrome c, aldolase, ovalbumin and bovine serum albumin) were recovered with an average yield of 73.6 +/- 2.3%. A membrane-bound protein, rat skeletal muscle Ca(2+)-ATPase (100 kilodalton) was also well recovered (over 60%). This method was applicable to the purification of proteins required for N-terminal amino acid sequencing and to raise antibodies.     

Sodium dodecyl sulfate-capillary gel electrophoresis of proteins using non-cross-linked polyacrylamide.

Proteins with relative molecular masses of 14,000 to 205,000 were separated by sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) using non-cross-linked linear polyacrylamide gels on both coated and uncoated fused-silica capillaries. It was determined that viscosity of the acrylamide solution was a major factor affecting column stability with linear acrylamide gels. When the viscosity of the acrylamide solution reaches 100 cP, electro-osmotically driven displacement of the gels is insignificant. Uncoated capillaries provided better resolution, stability, and reproducibility than surface coated capillaries when the concentration of linear polyacrylamide was greater than 4%. At lower gel concentrations, non-cross-linked polyacrylamide is easily displaced from the columns. A calibration plot of log molecular mass vs. mobility with non-linear polyacrylamide was linear, which indicated that resolution was equivalent to that obtained with cross-linked acrylamide. Separations with model proteins indicated that baseline resolution between protein species that vary 10% in molecular mass can be achieved.     

An improved sodium dodecyl sulfate-polyacrylamide gel electrophoresis system for the analysis of membrane protein complexes

Membrane protein complexes such as the reaction center complexes of oxygenic photosynthesis or the complex I of mitochondira are composed of many subunit polypeptides. To analyze their polypeptide compositions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a wide range of molecular sizes has to be resolved, especially in the low molecular mass range. We have improved the traditional Tris/HCI buffer systems adopting a Tris/2-(N-morpholino)ethanesulfonic acid (MES) buffer system containing 6 M urea. This gel system was used with an 18-24% acrylamide gradient for the separation of polypeptides with molecular masses from below 5 kDa to over 100 kDa. This buffer system can also be applied to the usual uniform concentration of acrylamide gel and also to minislab gels.     

Mass spectrometric analysis of blotted proteins after gel electrophoretic separation by matrix-assisted laser desorption/ionization.

The molecular masses of electroblotted proteins were determined with a time-of-flight mass spectrometer by matrix-assisted laser desorption/ionization directly from blot membranes. Therefore standard proteins, separated by polyacrylamide gel electrophoresis, were electroblotted onto polyvinylidene difluoride or polyamide membranes by standard procedures. Pieces of membrane containing the protein of interest were soaked in matrix solution and analyzed in the mass spectrometer.     

Use of chromatofocusing for separation of beta-lactamases. IX. Analytical chromatofocusing for the separation of a chromosomal cephalosporinase from Proteus vulgaris 1028

Simultaneous purification and isoelectric point (pI) determination was carried out at analytical scale of the chromosomal cephalosporinase from the Proteus vulgaris 1028 strain. Comparison of the enzyme to the purification results with m-aminophenylboronic acid-agarose affinity chromatography with sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed that minute amounts of accompanying proteins having identical pI values but different molecular masses were found in the chromatofocused preparation. The molecular mass of the enzyme was 24,000 dalton. The pI was found to be 8.3.     

Analysis of immune complexes of cerebrospinal fluid by two-dimensional gel electrophoresis

From the cerebrospinal fluid of 32 patients with different neurological diseases immune complexes were isolated using protein A-Sepharose. The isolated heavy and light chains and their constituents were analyzed by two-dimensional gel electrophoresis. In addition to immunoglobulins, some proteins such as albumin, apolipoprotein A-I and a number of unknown proteins were detected in all preparations. A complex consisting of three proteins with molecular masses between 52-55 kDa reacted slightly with polyclonal antibodies to glial fibrillary acidic protein. Whether the linkage between these antigens and the Ig is due to the Fab region or the Fc region remains unknown in our study. In some immune complexes of neurological diseases such as amyotrophic lateral sclerosis, astrocytoma and multiple sclerosis, differences are easily recognizable in the gel pattern.     

Size-based separations of proteins by capillary electrophoresis using linear polyacrylamide as a sieving medium: model studies and analysis of cider proteins

Electrophoretic conditions to separate sodium dodecyl sulfate (SDS)-protein complexes according to their relative molecular mass by capillary electrophoresis (CE) using linear polyacrylamide as a sieving matrix were examined. Five purified proteins with relative molecular masses between 14 400 and 66 200 Da were separated on a coated fused-silica capillary with an internal diameter of 100 microm and an effective length of 24 cm (total length, 32.5 cm). Benzoic acid was added to the solution of purified proteins as internal standard; beta-mercaptoethanol was also added as reducing agent. The running buffer composition was 0.05 M tris(hydroxymethyl)aminomethane (Tris), 0.035 M aspartic acid, 0.1% m/v SDS, 4% m/v acrylamide, the resulting pH being 8.0. The applied voltage was 7 kV (reversed voltage polarity) in order to avoid high current intensities. Under optimized conditions, the five proteins were separated in less than 15 min, with a % relative standard deviation (RSD) between 0.2 and 0.4 for migration times in the same day. Good efficiency (values between 150 000 and 40 000 N/m) and resolution (values between 2 and 2.8) were obtained. The inverse of relative migration times was found to correlate with the logarithm of their relative molecular mass. Finally, cider proteins were analyzed and their relative molecular masses were determined. These results were compared with those obtained by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).     

3D打印便携式凝胶电泳装置用于蛋白质的快速检测

随着现场分析对于快速、便携和经济型检测的需求,分析仪器的便携化和微型化备受关注。3D打印技术的不断发展,将会极大推动小型化、便携式实验设备的开发和研制。分析仪器的微型化有助于促进资源不足地区在医疗现场、食品安全和环境污染等方面的现场监测。目前,用于蛋白质分离的凝胶电泳装置多为实验室用小型化分析仪器,可用于现场快速分离蛋白质的小型化仪器尚未见报道。该研究设计加工了一款便携式凝胶电泳装置,用于蛋白质的快速分离检测。首先,通过3D打印加工的凝胶电泳装置可在实验室内方便、快捷、低成本的复制。其次,通过对预染蛋白质相对分子质量标准的分离测试,对该系统结构进行优化。优化后该凝胶电泳装置电泳槽的尺寸仅为15 mm×20 mm×17 mm,采用3D打印技术可在5 h内加工完成,耗费打印材料10 mL。正负极所用电泳缓冲液共需4 mL,所使用的25 V锂电池可实现100 h左右的工作时间。装置优化后可实现蛋白质的快速高效分离。随后,在5种常用蛋白质相对分子质量标准的分离中,该装置与商业化平板凝胶电泳分离效果相当,同时具备更快的分离速度。该研究在便携式凝胶电泳装置的开发及其在蛋白质快速分离方面取得了初步成果,但在分离完成后立即对蛋白质进行定量分析以及更多实际样品的应用方面还需要进一步研究。     

Virtual two-dimensional gel electrophoresis of high-density lipoproteins

High-density lipoproteins (HDLs) isolated by immunoaffinity chromatography and separated by immobilized pH gradient-isoelectric focusing (IPG-IEF) were examined by mass spectrometry directly, applying a new proteomics technology, virtual two-dimensional (2-D) gel electrophoresis. A preliminary examination of HDL particles has revealed at least 42 unique masses for protein species with isoelectric points between pH 5.47-5.04, some of which have not been observed previously. By delivering masses of intact proteins from complex cellular mixtures in a format that correlates directly to classical 2-D gel analyses, virtual 2-D gel electrophoresis constitutes a general discovery tool to expose and monitor protein isoforms and post-translational modifications. Furthermore, its general ability to deliver ions from sub-picomole level proteins enmeshed in complex cellular mixtures potentially fulfills the need of top-down proteomics to obtain intact protein ions from microscale samples. Additional comparison of such data to 2-D gel analyses and their identified proteins may elucidate the functions of the individual apolipoprotein components and the cardioprotective effects of HDL.     

Visualization and analysis of molecular scanner peptide mass spectra

The molecular scanner combines protein separation using gel electrophoresis with peptide mass fingerprinting (PMF) techniques to identify proteins in a highly automated manner. Proteins separated in a 2-dimensional polyacrylamide gel (2-D PAGE) are digested in parallel and transferred onto a membrane keeping their relative positions. The membrane is then sprayed with a matrix and inserted into a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer, which measures a peptide mass fingerprint at each site on the scanned grid. First, visualization of PMF data allows surveying all fingerprints at once and provides very useful information on the presence of chemical noise. Chemical noise is shown to be a potential source for erroneous identifications and is therefore purged from the mass fingerprints. Then, the correlation between neighboring spectra is used to recalibrate the peptide masses. Finally, a method that clusters peptide masses according to the similarity of the spatial distributions of their signal intensities is presented. This method allows discarding many of the false positives that usually go along with PMF identifications and allows identifying many weakly expressed proteins present in the gel.     

Enzymatically modified Soy Protein

Optimum temperature and pH for the isolation of soy protein isolate (SPI) from soy protein concentrate (SPC) were established. Enzymatic hydrolysis of SPI with enzymes of different specificities such as trypsin, chymotrypsin, papain and urease was carried out and the products of hydrolysis were characterized by molecular mass determination [sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)] and thermal techniques [differential scanning calorimetry (DSC) and thermogravimetric analysis (TG)]. Enzymatic hydrolysis resulted in a significant reduction in molecular masses. However the thermal stability of hydrolysed SPI was similar to native SPI indicating that it is independent of molecular mass. DSC studies indicated an increase in temperatures of endothermic transition associated with SPI denaturation and loss of absorbed moisture in samples of lower molecular masses. This revised version was published online in August 2006 with corrections to the Cover Date.     

PHOTORECEPTOR PROTEINS AND PIGMENTS IN THE PARAFLAGELLAR BODY OF THE FLAGELLATE Euglena gracilis

Abstract— –The presumed photoreceptor for phototaxis, the paraflagellar body, in the flagellate Euglena gracilis , was isolated still attached to the flagellum. After solubilization, fast protein liquid chromatography (FPLC) analysis yielded four major protein fractions with the chromophoric groups still attached. Fluorescence spectra showed that three fractions had excitation peaks at 380 nm and emission peaks around 450 nm indicative of pterins, while the fourth chromoprotein had a fluorescence emission at 520 nm and an excitation peak at 450 nm, indicative of a flavin. The separated proteins were analyzed by gel electrophoresis: the pterin binding proteins have apparent molecular masses between 27 000 and 31 600 and the flavin binding protein has an apparent molecular mass of 33 500.     

Differences between predicted and observed sequences in Saccharomyces cerevisiae

We recently studied the protein composition of a Saccharomyces cerevisiae wine yeast strain (K310) of enological interest. About 2,500 spots of 8-250 kDa observed molecular mass were resolved by two-dimensional gel electrophoresis. Experimental molecular masses and isoelectric points were calculated for most of them. Twenty-seven proteins were subjected to Edman microsequencing. N-terminal sequences of 12/27 proteins were determined, whereas internal sequences of 6/27 proteins were obtained following in situ proteolysis. Comparison between the experimental data and those reported in the SWISS-PROT database revealed some differences between genotypic and phenotypic sequences. These are indicative of the changes a protein can undergo with respect to the primary structure coded by the genomic DNA. Our results highlight the need to complement genomic analysis with detailed proteomics in order to refine the vast amount of information provided by DNA sequencing and to find an exact correlation between genome and proteome.     

Automated protein analysis by online detection of laser-induced fluorescence in slab gels and 3-D geometry gels

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) still remains the most reliable and comprehensive analytical method for the evaluation of protein extracts. However, conventional SDS-PAGE is time-consuming and, thus, unpractical if several tens or hundreds of samples must be examined. We show that SDS-PAGE protein analysis can be automated using slab gel DNA sequencers and compare the instrument's performance with conventional SDS-PAGE in terms of resolution, sensitivity and sample capacity. Labeled protein bands are detected online by laser-induced fluorescence (LIF) and the acquired signals are electronically stored for further processing, avoiding gel staining and scanning. Appropriate software allows immediate display of recorded data and convenient evaluation. The method provides a higher sensitivity and dynamic range than conventional Coomassie-stained gels and the resolution of proteins with different masses is independent of the polyacrylamide concentration. Internal markers can also be used for direct quantification and assignment of the molecular masses. Additionally, we present a novel electrophoresis instrument for the simultaneous separation and online LIF detection of all samples of a microtiterplate in parallel lanes in a 3-D geometry gel cylinder. The specific gel thermostatting concept prevents irregular sample migration (smiling) and improves the reproducibility and comparability of individual separation patterns. In combination with the expected large capacity of 384 or 1,536 samples, this makes the instrument a valuable tool for high-throughput comparative screening applications.     

Information on DNA conformation derived from transverse pore gradient gel electrophoresis in conjunction with an advanced data analysis applied to capillary electrophoresis in polymer media.

Abnormally slow migration of DNA is conventionally viewed as being due to an abnormal conformation relative to "linear" standards. The evidence for this rests on a few instances where nonlinear DNA structures have been established by independent methods and yield low mobilities relative to standards. Transverse pore gradient gel electrophoresis of authentically bent kinetoplast DNA and of an upstream activator sequence (UAS) of an E. coli operon promoter shows in addition that curves of migration distance vs. gel concentration ("Ferguson curves") of such abnormally conformed DNA differ from those of "linear" standards. Since Ferguson curves are interpretable with regard to molecular size in concordance with a mathematical model (Ogston model), transverse pore gradient gel electrophoresis provides a simple means of correlating abnormally slow migration of DNA with molecular size. In addition, transverse pore gradient gel electrophoresis is able to distinguish between DNA banding which exhibits a steeper dependence on gel concentration than "linear" standards from one which shows the same dependence. The former appears characteristic of circularly bent DNA and gives rise to a substantial retardation, the latter of bending across a knot or kink in the DNA chain associated with a relatively minor retardation relative to standards. Circularly bent restriction fragments formed from kinetoplast DNA retain the characteristic intersecting Ferguson curves on the transverse pore gradient gel. Another authentically "abnormal" DNA structure recognizable on transverse pore gradient gels is supercoiled DNA derived from the reaction of topoisomerase with a plasmid. Different lengths of supercoiled sequences give rise to parallel Ferguson curves clearly intersecting with those of linear standards.(ABSTRACT TRUNCATED AT 250 WORDS)     

A short history of electrophoretic methods

High resolution separation of proteins, based on charge differences, is possible with disc electrophoresis, displacement electrophoresis (isotachophoresis) and notably isoelectric focusing (IEF). Size separation is obtained in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The combination of gel IEF, followed by SDS-PAGE in a second-dimensional slab gel, i.e. two-dimensional gel electrophoresis, affords the highest resolution with up to several thousand spots per gel. Staining of proteins gives high resolution patterns which can be scanned and stored in comprehensive databases. Over the last 10 years the electrophoretic separation in gels and subsequent visualization of nucleic acids (DNA, RNA) and even genes as well as nucleotides have been much improved, making possible efficient mapping of the genes in humans and all other organisms. This has led to the biggest concerted endeavor in the history of science, i.e. the mapping of the human genome, which will be of importance as long as mankind exists. In the last years electrophoresis in capillaries has attracted much interest because for numerous substances, such as proteins nucleic acids, pharmaceuticals, metabolites, and peptides, it offers high resolution on the analytical scale with over 1 million theoretical plates. Electrophoretic methods have unprecedented impact on life sciences, providing a basis for unique advances in biochemistry, molecular biology, genetics, gene technology and medicine.     

Proteome analysis of activated murine B-lymphocytes

Proteins extracted from murine B-lymphocytes after in vitro stimulation by lipopolysaccharide were separated by two-dimensional (2-D) polyacrylamide gel electrophoresis and analyzed by matrix assisted laser desorption/ionization mass spectrometry. Structural information on the protein entities from 153 spots was obtained. Since many of these spots occur as members of spot families, a smaller number --98 genes-- was found to be coding for the identified spots. The elucidated proteins belong to groups of functional categories; we found 26 enzymes, 36 regulatory proteins, 15 chaperones, 15 structural proteins, 4 immunoglobulins, 1 ribosomal and 1 histone protein. A comparison between expected and observed molecular masses yields a good correlation for the majority of the compared spot entities. This set of proteins now identified in the context of a lymphocyte 2-D gel pattern should advance further studies on lymphocyte functions.