Detection of turnip crinkle virus on agarose gel electropherograms at the nanogram load level

The previous conditions for the physical characterization of turnip crinkle virus (TCV) by quantitative agarose gel electrophoresis [1, 2] were limiting the method to the microgram load level and were therefore insufficiently sensitive to satisfy the need in many areas of virology for detection of viruses containing single-stranded RNA at the nanogram level. The present report remedies that defect by presenting a technique compatible with the nanogram load level of such viruses. The technique is based on a reduction of gel thickness and on the use of silver staining.     

Resolution of 8-aminonaphthalene-1,3,6-trisulfonic acid-labeled glucose oligomers in polyacrylamide gel electrophoresis at low gel concentration

A discontinuous Tris-Cl/acetate (OAc) buffer system, unprecedently containing OAc as the trailing constituent, and operative in polyacrylamide gel electrophoresis (PAGE) at low polyacrylamide concentration (T = 4.8%) is described in the paper. The characteristics of the electrophoretic system are illustrated by the resolution of fluorescent 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS)-labeled malto-oligosaccharides and dextran homopolymers. In this buffer system, the resolving phase is constituted by Tris-OAc behind a moving boundary formed between the leading chloride ion of Tris-HCl gel buffer and the trailing OAc ion provided by a catholyte of NH(4)OAc. In contrast with the results obtained with Tris-CI/glycinate buffer commonly used in electrophoresis, or with Tris-CI/borate, the best resolution of the glucose oligomers containing 1-4 glucose units in Tris-OAc, pH 8.8, ionic strength of 0.08, was obtained at 4.8% polyacrylamide concentration, using 0.5 M NH(4)OAc, pH 9.5 as the catholyte. Under those conditions, the ANTS-glucose oligomers were separated with mobilities decreasing from glucose to maltohexaose. The linear Ferguson plots (log relative mobility, R(f), vs.%T) of the glucose oligomers show that the surface net charge of those oligomers is inversely related to their sizes, given by the slopes, K(R), of the plots. The molecular weight of the oligomers is directly but nonlinearly related to K(R). The novel electrophoretic system illustrated here for separation of short ANTS-saccharides can be potentially applied to the resolution of other biomolecules such as rapidly migrating DNA, peptides or proteins.     

Discontinuous native protein gel electrophoresis

Analysis of the oligomeric state of a native protein usually requires analytical ultracentrifugation or repeated gel filtration to calculate the protein's size. We have developed a discontinuous native protein gel electrophoresis system that allows the separation of even basic proteins according to their size, oligomeric state, and shape. This gel system combines the addition of negative charges to the proteins by Serva Blue G with a discontinuous buffer system and gradient gels. As in SDS-PAGE, chloride constitutes the high mobility anion in the gel and anode buffer. However, for sample focusing this system employs histidine instead of glycine as the slow dipolar ion following from the cathode buffer to improve migration of basic proteins. In addition, proteins run into gel pores corresponding to their size and shape in the gradient gel. Using this gel system, we show that the polypyrimidine tract-binding protein (PTB) is a monomer.     

Discontinuous buffer system for polyacrylamide and agarose gel electrophoresis of DNA fragments

DNA fragments up to 9 kb in size were stacked and separated by polyacrylamide gel electrophoresis, and those up to 50 kb in size by agarose gel electrophoresis, using a discontinuous buffer system. Polyacrylamide gels at pH 8.9, 2 degrees C, 0.01 M ionic strength, yielded sharp bands with DNA loads of 8 micrograms/cm2 of gel of a mixture of 19 DNA fragments in the size range of 72-23130 bp, while agarose gels at pH 8.5, 25 degrees C, provided well-resolved, unperturbed bands at 0.04 M ionic strength with DNA loads of 1 microgram/cm2 of the same mixture. Note that the ionic strength of the agarose gels is comparable to the conventionally used 0.5 x TBE (Tris-borate-EDTA) buffer, while that successfully applied to polyacrylamide is seven-fold less than the ionic strength of conventionally used 1 x TBE buffer, with a substantially shorter duration of electrophoresis as a result. The application of a discontinuous buffer system to the gel electrophoresis of DNA results in (i) Band identification by Rf, the migration distance relative to a sharply defined "buffer front" (moving boundary). This is sufficiently labor saving, compared to determining absolute mobilities, so as to render practical the expression of bands as numbers, with benefits for data storage, statistical manipulations and physico-chemical exploitation of mobility data. The use of Rf's also circumvents loss of precision in mobility measurement resulting from progressive band spreading of dye bands used as a front. (ii) A uniformly and highly concentrated starting zone, beneficial to resolution, is obtained, without the losses by which separate concentration steps are usually burdened.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of DNA restriction fragments and polymerase chain reaction products towards detection of the AIDS (HIV-1) virus in blood.

A high-performance capillary electrophoresis system with a polysiloxane-coated capillary and polymeric buffer additives was investigated for the analysis of DNA restriction fragments and polymerase chain reaction (PCR) products. Mobility data and Ferguson plots of the DNA fragments at different polymer (hydroxypropylmethylcellulose) concentrations indicated that effective molecular sieving was obtained consistent with existing data of conventional gel electrophoresis and with recent HPCE data. The precision and peak efficiency were excellent and the system was applied to the analysis of specific co-amplified DNA sequences (HIV-1 and HLA-DQ-alpha). After PCR, ultrafiltration was used in the sample preparation step to desalt the sample and to remove superfluous PCR reaction products. Electrokinetic injection was used for sample introduction into the capillary. The addition of ethidium bromide to the buffer resulted in longer migration times of DNA fragments and better peak resolution. During HPCE, an artifact associated with dilute DNA solutions leading to the appearance of extra peaks in the electropherogram was found.     

Electrophoresis in one buffer at two pH values

A theory for discontinuous electrophoresis in a polyacrylamide gel is presented for one buffer at two pH values. It is shown that polyions stack between identical leading and trailing ions, and resolve in a gel of constant polyacrylamide concentration. The theory is illustrated by the separation of serum protein polyions in a Tris-glycinate buffer of pH 8.19 in the well-forming gel, and pH 9.16 in the resolving gel. The selected concentrations and electrolyte ionization degrees of Tris and glycine have values at which the serum protein polyions stack between the resolving and electrode buffers, followed by separation in the resolving gel.     

High resolution electrophoresis of soybean seed isoesterases

A modified discontinuous buffer system for vertical polyacrylamide electrophoresis was developed with large changes in component concentration from the original Tris-HCl/Tris-glycine system of the Ornstein-Davis type. The new buffer system is able to resolve the fast soybean seed isoesterases crucial for cultivar discrimination. A higher number of anodic bands is obtained in comparison with separations under standard conditions. A "gelating boundary", resulting from the aggregation of seed proteins, is retarded and does not migrate out of the upper stacking gel. The same buffer modification is also beneficial to the separation of native seed proteins.     

Program in BASIC for Ferguson plot analysis, using a personal computer: application to gel electrophoresis in a continuous buffer

A program in BASIC suitable for personal computers is described which is applicable to gel electrophoresis conducted in a single (continuous) buffer. The curve fitting is to a polynomial function, allowing for an objective selection of the most appropriate curve type and order--linear, convex or concave--in the particular application. Results do not differ significantly from previous programs for evaluation of linear Ferguson plots or of curve fitting to an exponential function for evaluating convex plots, executed on mainframe computers such as the DEC-10 (Digital) and IBM 370 computers. Thus, the program combines original versatility with, for the first time, the possibility for widespread application of Ferguson plot analysis on personal computers.     

Hybrid microdevice electrophoresis of peptides, proteins, DNA, viruses, and bacteria in various separation media, using UV-detection

Recently we described the design of a hybrid microdevice for micro(nano)electrophoresis and electrochromatography, discussed its advantages and disadvantages compared to conventional microdevices and presented a few applications with low-molecular-weight samples. In this paper, we demonstrate the broad application range of this device using UV-based analyses of (i) peptides by free-zone electrophoresis and electrophoresis in a recently introduced gel (polyacrylamide cross-linked with allyl-beta-cyclodextrin), (ii) proteins by electrophoretic molecular-sieving in a polymer solution supplemented with SDS, (iii) DNA fragments by electrophoresis in the above gel, (iv) virus particles in this gel, as well as in free buffer and (v) bacteria in free buffer. To illustrate the advantages of the hybrid microdevice we can mention that electrophoresis of proteins in a polymer-containing buffer, supplemented with sodium dodecyl sulfate (SDS), in a 4.30 (2.75) cm long channel gave a resolution similar to that in conventional capillary electrophoresis in a 23.5 (18.6) cm long capillary and analysis times which were 15-fold shorter.     

Free mobility determination by electrophoresis in polyacrylamide containing agarose at a nonrestrictive concentration

In the determination of the free mobility, related to the surface net charge, by quantitative gel electrophoresis, the previous arbitrary extrapolation of Ferguson plots from the lowest gel concentrations that give a mechanically stable gel to 0% T has recently been replaced by measurement of mobilities across that concentration range, using the addition of 0.5% agarose to polyacrylamide at the various low concentrations in application to a DNA fragment 155 bp in size (Orbán, L. et al., in preparation). The present study applies that approach to several proteins and DNA fragments smaller than 1300 bp, using 0.4% agarose in polyacrylamide gels of varying concentration. The intercepts of the plots with the mobility axis provide experimental data by which the free mobility in polyacrylamide gel electrophoresis can be estimated for molecules not significantly retarded in their migration at the agarose concentration admixed to polyacrylamide. Across the gel concentration range below 3% T, in the presence of agarose, the Ferguson plots of proteins and DNA fragments are convex. It was shown by mass spectrometry that this convex curvature of the plots in the mixed polymer is not significantly due to low polymerization efficiency in the concentration range of liquid polyacrylamide (below 3%T).     

Electrophoretic techniques in element species analysis. Comparison between flow-through and flat-bed techniques

A comparison of electrophoretic techniques for the separation of metal-protein complexes from food extracts is described. A preparative flow-through electrophoresis system with continuous elution of the fractionated substances and an analytical flat-bed gel electrophoresis technique with off-line electro-elution are optimized with regard to this separation problem. The metal-protein complexes are extracted from four flour samples using Tris-glycine buffer (pH 8.3). For the separation, polyacrylamide gels of 14% T and 3% C are prepared and Tris-HCl (pH 8.9) is used as an electrophoresis buffer. For a soy bean flour, not only the separation of protein fractions is achieved but also the metal distribution patterns that are determined by flame atomic absorption spectrometry are given. The results show that the use of the flow-through technique is limited to special fields of application, whereas the flat-bed electrophoresis with subsequent electro-elution of metal-protein complexes is a useful technique in element species analysis.     

Analytical and micropreparative separation of peptides by capillary zone electrophoresis using discontinuous buffer systems.

The tiny injection volumes that are usually necessary to maintain the high efficiency of capillary zone electrophoresis present a major problem if only limited sample amounts are available. To increase the sample load, discontinuous buffer systems were developed that allow the on-column concentration of dilute samples. Injection volumes can be increased in this way by at least a factor of 30. These stacking systems were applied to the analysis of tryptic peptides, to the purity checking of high-performance liquid chromatographic fractions and for the micropreparative separation of peptides with subsequent amino acid sequence analysis.     

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.     

Integration of solid-phase extraction membranes for sample multiplexing: application to rapid protein identification from gel-isolated protein extracts

The present report describes the design and application of a dual sprayer system for high-throughput proteome analysis. This system comprises parallel solid-phase extraction cartridges used for preconcentration and desalting of proteolytic digests prior to nanoelectrospray mass spectrometry analyses. Tryptic peptides from in-gel digest of protein bands/spots are first adsorbed on styrene divinyl benzene membrane and subsequently eluted with a short plug of organic buffer prior to infusion to the mass spectrometer at a flow rate of typically 500 nL/min. Tryptic peptide eluting from the membrane are analyzed by the mass spectrometer by moving in turn each sprayer in front of the sampling orifice. Sequential injection, preconcentration and analyses of tryptic digests are typically achieved with a throughput of up to 3.5 min/sample and a detection limit of approximately 8-80 fmol per injection. Replicate injections of peptide mixtures indicated that reproducibility of peak areas ranged from relative standard deviations (RSD) of 1.1% to 4.5%. The application of this device is demonstrated for digests of gel-isolated proteins obtained from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation of rat liver plasma membrane and from two-dimensional gel electrophoresis of total cell lysate extracts from human prostatic cancer cell.     

Concave Ferguson plots of DNA fragments and convex Ferguson plots of bacteriophages: evaluation of molecular and fiber properties, using desktop computers.

A desktop computer program evaluating physical properties of DNA and bacteriophages is presented. The analysis is based on data obtained from capillary and submarine-type agarose electrophoresis. Native molecular/particle properties and properties of the gel (or polymer) medium can be derived from electrophoresis at several gel concentrations. This is done conveniently by a computerized evaluation of the semi-logarithmic plot of mobility vs. gel concentration, designated the Ferguson plot. In application to most proteins, this plot is linear and computer programs exist to evaluate it. However, nonlinear Ferguson plots have assumed great importance in view of the fact that the plots are concave for DNA. Similarly, convex plots are important since they prevail in the electrophoresis of large particles in agarose. The computer program reported here is the first to (i) address concave Ferguson plots and (ii) allow for the evaluation of both cases using a desktop computer. Program ELPHOFIT version 2.0, a Macintosh application, is available upon request.     

Electrophoresis and detection of tin-labeled DNAs on open-faced gels.

Alternative protocols are necessary for the use of polyacrylamide gel electrophoresis in genome scale sequencing and mapping studies. The use of radioisotopes and manual gel reading will have to be replaced with a flexible labeling system that can be detected at levels similar or to better than radioisotopes but allows automated, high-speed detection. Labeling with stable isotopes is such an alternative. These nondecaying isotopes have the potential to be detected in sub-attomole quantities, despite being surrounded by the gel matrix, due to the high selectivity and sensitivity of resonance-ionization spectroscopy coupled with a mass spectrometer. In this study the detection limits of sputter-initiated resonance ionization spectroscopy (SIRIS) are investigated using thin, open-faced polyacrylamide gels supported by plastic. This system allows reproducibility and flexibility in the choice of gel size and buffer system since the gel can be cast, washed free of polymerization by-products, dried, and stored until use. Various concentrations of an Sn-labeled oligomer were run on these gels and loads of 5 femtomoles/mm could be detected on a 240 microns thick gel. Gels as thin as 60 microns lower the detectable concentration loads to 1 femtomole/mm. The limiting factor is tin contamination in the gel which, if reduced, will further increase detection. Polymerase chain reaction (PCR) products can also be labeled and detected using Sn isotopes, which could prove useful in mapping studies. Also presented are techniques which will facilitate resolution of these PCR products on open-faced gels by employing discontinuous buffers systems and DNA mobility modifiers.     

A versatile electrophoresis system for the analysis of high- and low-molecular-weight proteins

A new, versatile, multiphasic buffer system for high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins in the relative molecular weight range of 300 000-3000 Da is described. The system, based on the theory of multiphasic zone electrophoresis, allows complete stacking and destacking of proteins in the above M(r) range. The buffer system uses taurine and chloride as trailing and leading ion, respectively, and Tris, at a pH close to its pK(a), as the buffering counterion. Coupled with limited variation in the acrylamide concentration, this electrophoresis system allows to tailor the resolution in the 6-200 kDa M(r) range, with minimal difficulties in the post electrophoretic identification processes.     

The electric field dependence of DNA mobilities in agarose gels: a reinvestigation

The electric field dependence of the electrophoretic mobility of linear DNA fragments in agarose gels was reinvestigated in order to correct the observed mobilities for the different temperatures actually present in the gel during electrophoresis in different electric field gradients. When corrected to a common temperature, the electrophoretic mobilities of DNA fragments less than or equal to 1 kilobase pairs (kbp) in size were independent of electric field strength at all field strengths from 0.6 to 4.6 V/cm if the gels contained less than or equal to 1.4% agarose. The mobilities of larger DNA fragments increased approximately linearly with electric field strength. If the agarose concentration was higher than 2%, the mobilities of all DNA fragments increased with increasing electric field strength. The electric field dependence of the mobility was larger in gels cast and run in Tris-borate buffer (TBE) than in gels cast and run in Tris-acetate buffer (TAE), and was more pronounced in gels without ethidium bromide incorporated in the matrix. Ferguson plots were constructed for the various DNA fragments, both with and without extrapolating the temperature-corrected mobilities to zero electric field strength. Linear Ferguson plots were obtained for all fragments less than or equal to 12 kbp in size in agarose gels less than or equal to 1.4% in concentration if the mobilities were first extrapolated to zero electric field strength. Concave upward curvature of the Ferguson plots was observed for DNA fragments greater than or equal to 2 kbp in size at finite electric field strengths. Convex downward curvature of the Ferguson plots was observed for DNA fragments greater than or equal to 1 kbp in size in agarose gels greater than or equal to 2% in concentration. The mobilities of the various DNA fragments, extrapolated to zero agarose concentration and zero electric field strength, decreased with increasing DNA molecular weight; extrapolating to zero molecular weight gave an "intrinsic" DNA mobility of 2.7 x 10(-4) cm2/Vs at 20 degrees C. The pore sizes of LE agarose gels cast and run in TAE and TBE buffers were estimated from the mobility of the DNA fragments.(ABSTRACT TRUNCATED AT 400 WORDS)     

Reversed-phase high-performance liquid chromatographic, gel electrophoretic and size exclusion chromatographic studies of subunit structure of arachin and its molecular species

Arachin and its molecular species (arachin I and arachin II) were separated and isolated. The number and kind of subunits of arachin, arachin I and arachin II were determined. Studies were carried out under different experimental conditions using slab gel electrophoresis, size-exclusion chromatography and reversed-phase high-performance liquid chromatography. Gel electrophoresis was done under varying concentrations of resolving gel. Tube gel as well as slab gel electrophoresis were used and continuous as well as discontinuous buffer systems were used for both types of electrophoresis. In addition, the subunits were separated by reversed-phase HPLC using a gradient program. Arachin and arachin II were found to have 12 subunits each while arachin I showed six subunits. The subunits of arachin I were allowed to reconstitute by removing SDS. Eight combinations were tried for studying the reconstitution pattern. Molecular weight and weight ratio in each case were also determined.     

Chiral separation of ephedrine isomers by capillary electrophoresis using bovine serum albumin as a buffer additive

A method utilizing bovine serum albumin (BSA) as buffer additive for chiral separation by means of capillary electrophoresis is described. Parameters that affect chiral separation, such as buffer pH, buffer concentration, BSA concentration, and organic modifier, are investigated. Baseline resolution of ephedrine-pseudoephedrine and norephedrine-norpseudoephedrine isomers are achieved in an uncoated capillary with a 20 mmol/L phosphate buffer at pH 9.0 in the presence of 10 micromol/L BSA and 15% (v/v) 2-propanol at 25 degrees C. The developed method can be applied for the analysis of ephedra plant extracts that contain the four test drugs.