Slab Electrophoresis Research Articles

Analysis of new charge-neutral DNA/RNA analogues phosphoryl guanidine oligonucleotides (PGO) by gel electrophoresis

Analysis and isolation of new charge-neutral phosphoryl guanidine oligonucleotides (PGO) by vertical slab electrophoresis were tested at different pH values (3–11) or in the presence of SDS as a micelle-forming agent. The most convenient way to analyze and purify phosphoryl guanidine oligonucleotides was by denaturing PAGE (8 M urea) at pH 3. The mobility of PGO is dependent on their A + C content. To analyze PGO containing only G, T or U, denaturing PAGE at pH 11 can be used, although the conditions need to be optimized. Bands were visualized by UV shadowing or Coomassie Brilliant Blue staining.

Separation of Lipase from Karnel of Pistacia khinjuke and Determination of it's Affinity Toward some Inhibitors in Mice In vivo and In vitro

The study included the purification of lipase from Pistacia khinjuke by the application of the ammonium sulfate precipitation (70%), dialysis, ion exchange chromatography and finally slab electrophoresis techniques. One isoenzyme was obtained having a molecular weight of 43597 dalton and specific activity of 3-10× 152 unit / mg protein. The maximum lipase activity was obtained at pH 7.5, temperature 40 C. The maximum velocity (Vmax) was 0.142 unit/ml/min and Km value was 0.666 mM. The type and ability inhibition of lipase was tested in presence of quercitine rutinoside and melatonine. The inhibition type was noncompetitive and the inhibition constant Ki values for the two inhibitors were 1.025 and 1.006 mM, respectively. The lipase affinity of normal and induced diabetic mice serum toward these compounds was tested. The treatment of animals by a dose of 523.3 mg quercitine rutinoside / Kg of body weight showed a significant decrease in lipase activity, but an insignificant decrease with a dose of 8 mg melatonine / Kg of body weight. The treatment of animals by these compounds led to a significant decrease in glucose, total cholesterol and ratio of total cholesterol/HDL-C levels, but a significant increase in HDL-C in serum of normal and induced diabetic mice. The inhibitory effect of quercitine rutinoside and melatonine toward lipase and biochemical parameters' levels studied may help in using these compounds as natural inhibiting drugs.

Evaluation of a microfluidics-based platform and slab electrophoresis for determination of size, integrity and quantification of in vitro transcribed RNA used as a component in therapeutic drug manufacturing

Ribonucleic acid (RNA) is gaining utility as a key component of immunotherapeutics to transiently express antigens or to modulate endogenous gene expression for clinical applications. As a key ancillary component of clinical grade products, RNA requires a robust method for quality control. Here we evaluated the microfluidics based platform and slab electrophoresis for determination of integrity, concentration and size of four in vitro-transcribed RNA products with sizes of 1611, 808, 475 and 290 nucleotides (nts). Our data demonstrate that the Bioanalyzer can determine both size and integrity of the RNA, but the analysis suffers from a strong well position effect. For the RNAs tested, the integrity values obtained by the Bioanalyzer demonstrate a reverse correlation with the size of the molecule and are lower than those obtained using slab electrophoresis. Agarose gel electrophoresis produced the information on size of the RNA molecule with good precision, accuracy and reproducibility. We highlight observations which need to be taken into account when developing and qualifying a method of choice for assessment of in vitro-transcribed RNA using either approach.

Development of a simple ampholyte-free isoelectric focusing slab electrophoresis for protein fractionation

Sample preparation is often necessary to separate and concentrate various compounds prior to analysis of complex samples. In this regard, isoelectric focusing (IEF) is one of the best sample preparation methods. With this approach, however, carrier ampholytes have to be introduced into the samples, which may result in matrix interferences. In this paper, a simple ampholyte-free IEF free-flow electrophoresis design was developed for the separation of proteins. β-Lactoglobulin, hemoglobin, myoglobin and cytochrome c were selected as model analytes. The experimental design took advantage of the electrolysis-driven production of H + and OH − ions that migrated from the anode and cathode, respectively, establishing a pH gradient spanning from 2.3 to 8.9. The separation chamber was filled with silanized glass beads as a support medium. Dialysis membranes were mounted at the two sides of the separation chamber (made of glass slides) and sealed with 2% agarose gel. The separated proteins drained from the outlets of the separation chamber and could be successfully collected into small glass tubes. The focusing process was visually observed and the separation was confirmed by capillary isoelectric focusing (cIEF) with pI markers.

Interaction study between double-stranded DNA and berberine using capillary zone electrophoresis

Two non-self-complementary 17-mer double-stranded DNA (dsDNA) with four different central base pairs were designed to systematically investigate the binding affinity and sequence specificity of berberine with dsDNA by capillary zone electrophoresis (CZE). The data analysis with the Kenndler model proved only low affinity between dsDNA and berberine and suggested some weak binding preference of berberine for AATT-containing to GGCC-containing dsDNA. The binding constant, Ka, between berberine and dsDNA(AB) was about (1.0 +/- 0.7) x 10(3) M(-1). In addition, the separation of single-stranded DNA (ssDNA) from dsDNA under simple electrophoretic conditions enabled CZE to be a potentially alternative tool to check the extent of DNA annealing, which is usually done by the time-consuming and labor-intensive slab electrophoresis.

Preparation and characterization of PEGylated adducts of recombinant human tumor necrosis factor-α from Escherichia coli

This study was conducted to get an insight into monomethoxypolyethylene glycol (MPEG) modified recombinant human TNF-α (rhTNF-α) derived from gene cloning and expression. The purification and modification processes were integrated together to make the scale-up production of PEG-modified rhTNF-α practical. Capillary electrophoresis was demonstrated to be a highly efficient tool in the biochemical characterization of the PEGylated products compared to slab electrophoresis. The cytotoxicity of MPEG-modified rhTNF-α was studied in vitro towards L929 cell line and found to decrease gradually along with the increase in the reaction ratio between the activated MPEG and the native rhTNF-α. The MPEG-modified rhTNF-α was more resistant to proteinase degradation in vitro than the native. When MPEG chains were released partially from the MPEG-modified rhTNF-α by alkaline pretreatment, the cytotoxicity of the MPEG-modified rhTNF-α was enhanced, which was in contrast to the native. These results would be helpful to explain the disagreement between the high bioavailability of MPEG-modified TNF-α in vivo and its decreased cytotoxicity in vitro.

Effect of temperature on the separation of long DNA fragments in polymer solution

Electrophoresis of long DNA fragments in polymer solutions is still attractive when performed in short capillaries. Then the separations can be accomplished in minutes rather than hours as is usual in various slab electrophoresis techniques. In this paper we focused on the behavior of large DNA fragments in pulsed field capillary electrophoresis under various temperature conditions. The mobility dependence of fragments of λ-DNA single-cut mixture on various frequencies at three different temperatures showed that the antiresonance mobility minima are shifted to higher frequencies at higher temperatures. This interesting result is explained in terms of the geometration model of DNA motion.

High Throughput Sample Preparation using Ultrafiltration in Multiwell Plate Format. Post-Sequencing Cleanup of Dye Terminator Sequencing Reactions for Capillary Electrophoresis.

A unique new method is described showing the use of microfiltration and ultrafiltration membranes in conjunction with gel permeation in 96 well plates. Using this technique it is possible to clean up DNA sequencing reactions to a level of template DNA purity that makes high DNA loading onto capillary electrophoresis (CE) sequencers possible. Loadings as high as 10, 000 ng allow sequencing on CE to be equal in performance to gel slab electrophoresis. This technique replaces alcohol precipitation for sequencing reaction cleanup with an easily automated process taking 1/6 the time.

Electrophoresis of DNA in novel thermoreversible matrices.

We demonstrate the feasibility of temperature-sensitive polymers as novel matrices for capillary and slab electrophoresis of DNA. These matrices combine the case-of-use of agarose with resolution properties of polyacrylamide. Two classes of matrices are used: (i) aqueous suspensions of gel microspheres and (ii) solutions containing uncross-linked temperature-sensitive polymers. When heated, the viscosity of these solutions drops dramatically because of the phase transition behavior of these polymers, and therefore these formulations are easy to pour or load. Results are presented for separation of double-stranded DNA fragments (< 2000 bp) in capillary, tube, and slab electrophoresis. Preliminary results are also presented for separation of single-stranded sequencing fragments by capillary electrophoresis. In the tube format, good resolution was obtained for phi X174/HaeIII fragments. In the slab format, excellent resolution of double-stranded DNA was obtained, including simultaneous separation of a 10 bp ladder up to 150 base pairs. In the capillary format, pBR322/MspI fragments were completely resolved, and single-base resolution of sequencing fragments was obtained up to 150 bases. We believe that temperature-sensitive polymers represent a new and promising class of electrophoretic media.

Rh D/d genotyping by quantitative polymerase chain reaction and capillary zone electrophoresis.

A safe and reliable method for determining RhD type (positive or negative) and zygosity (D/D or D/d) could have applications, for instance, in the prediction of the D genotype of a father in couples where there is an RhD-negative woman at risk of fetal alloimmunization. Capillary zone electrophoresis (CZE) is proposed as a novel, reliable and powerful method for quantitative evaluation of polymerase chain reaction (PCR) products. RhD is determined by amplifying a 136 bp region common to the RhCcEe and RhD genes and a 186 bp region specific of the RhD gene. RhD positive and negative samples are identified by polyacrylamide gel slab electrophoresis, followed by silver staining of the DNA bands, and by CZE in sieving liquid polymers, with direct on-line peak densitometric evaluation by exploiting the intrinsic UV absorbance of the DNA fragments at 254 nm. The CZE method allows not only a reliable assessment of the RhD type (the presence of both 136 bp and 186 bp fragments indicating an RhD-positive type, the presence of only the 136 bp fragment indicating an RhD-negative type), but also a rapid determination of the zygosity based on the quantitative expression ratio of the 136 bp/186 bp pair. Thus, a 2:1 peak ratio clearly indicates a D/D homozygous individual, whereas a 3:1 peak ratio gives evidence of a D/d heterozygous individual.

Purification and some properties of a Vero toxin 2 (Shiga-like toxin II) variant (SLT-IIva) of Escherichia coli isolated from infantile diarrhea

A variant of Vero toxin 2 (VT2), or Shiga-like toxin II (SLT-II) was purified from a cloned strain of Escherichia coli carrying a gene that encodes SLT-IIva, which is most closely related to SLT-IIv (VT2vp, VTe) in its nucleotide sequence. The purified SLT-IIva showed a slight difference from the purified SLT-IIv (VT2vp) in mobility on polyacrylamide gel disc electrophoresis and SDS-polyacrylamide gel slab electrophoresis and in pl, but both showed a similar potency in biological activities. Ouchterlony double diffusion test revealed that the purified SLT-IIva and SLT-IIv (VT2vp) formed a spur against anti-SLT-IIva and anti-SLT-IIv (VT2vp) antisera.

Dissociation and electrophoretic separation of dextranase and dextranase inhibitor from a tightly bound enzyme-inhibitor complex of Streptococcus sobrinus.

Endodextranase was separated from dextranase inhibitor in culture filtrates of Streptococcus sobrinus by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) in gel slabs containing blue dextran. Sample preparation included dissociation of the enzyme from its inhibitor by boiling for 1 min in SDS. During subsequent incubation of the gel, dextranase was located as clear bands on a blue background, and dextranase inhibitor appeared as blue zones on a clear background following incubation in dextranase solution. The enzyme and the inhibitor existed in multiple forms, and the range of molecular masses for dextranase (223-132 kDa) permitted an excellent separation from dextranase inhibitor (49-25 kDa). Although dextranase-negative mutants, and wild type strains grown at low dilution rate in the chemostat, were devoid of free dextranase activity, the enzyme was easily located by analytical SDS-PAGE. Likewise, analysis of filtrates from wild type strains, which contained no free inhibitor activity when growth occurred at high dilution rate, revealed dextranase inhibitor activity on the gels. The total production (free + combined) of dextranase and inhibitor by S. sobrinus was determined by dissociation of enzyme-inhibitor complexes in concentrated cell-free filtrates, their separation by preparative SDS-PAGE and electroelution from the gels, followed by renaturation of protein activity. From a comparison of activity tests of free dextranase and free inhibitor in untreated filtrates with the results of similar tests on renatured electroeluates, the proportion of each constituent bound into a complex under each growth condition could be deduced.

5164065 Non-breakable, electrically insulating sample well inserts for slab electrophoresis: Gregory Bettencourt, George Fernwood, Efrem G Hernandez, Lin Crawforth assigned to Bio-Rad Laboratories Inc

5164065 Non-breakable, electrically insulating sample well inserts for slab electrophoresis: Gregory Bettencourt, George Fernwood, Efrem G Hernandez, Lin Crawforth assigned to Bio-Rad Laboratories Inc

Ultrasensitive plasmid mapping by high performance capillary electrophoresis.

This paper compares high performance capillary electrophoresis (HPCE) and conventional slab electrophoresis in mapping of four closely related plasmids with three different restriction enzymes. The plasmids express full length and truncated forms of a growth factor receptor oncogene product and were digested with HpaII, HaeIII and RsaI. The resulting oligonucleotide fragments were under 2000 base pairs in length, a size well suited to separation by HPCE with linear polyacrylamide as a sieving matrix. Plasmid mapping is an essential tool in biotechnology both for the design of an expression system and for monitoring the stability of the expression system during fermentation. HPCE can yield much higher resolution of oligonucleotides than attainable in conventional agarose gel electrophoretic procedures for plasmid mapping. In the examples described here, the HpaII digests provided the surest identification of individual plasmids in the HPCE analysis and could discriminate among all four plasmids. In conventional slab electrophoresis, however, the RsaI digests provided the best discrimination, although two of the plasmids in this system yielded essentially identical electrophoretic patterns. Hence the optimal restriction enzyme for plasmid mapping applications with HPCE may differ from that selected on the basis of conventional slab gel analysis, and the former technique can provide higher discrimination among related plasmids. The advantages of the HPCE format with respect to speed, low sample consumption and resolution are described.

Microcrystalline cellulose-hydrolyzing cellulase (endo-cellulase) from Trichoderma reesei CDU-11.

A microcrystalline cellulose (Avicel)-hydrolyzing cellulase (an endocellulase) was isolated from a preparation of Trichoderma reesei CDU-11 and purified successively by DEAE-Sepharose CL 6B and Concanavalin A-Sepharose column chromatography, and preparative electrophoresis and on Sephadex G-100 gel filtration. The cellulase preparation showed a single protein band on SDS-PAGE slab and gel disc electrophoresis. Its molecular weight was estimated to be 64, 000 by Sephadex G-100 gel filtration and 67, 000 by SDS slab electrophoresis. The isoelectric point was at pH 4.75 on polyacrylamide gel electric focusing. This cellulase hydrolyzed more specifically water insoluble microcrystalline cellulose such as Avicel rather than water soluble cellulose such as carboxymethyl-cellulose. It produced glucose, cellobiose and cellotriose from cellotetraose and cellohexaose, and it produced a significant amount of glucose and cellobiose from Avicel. Based on these results, it appears that the cellulase should be regarded as an endo-type cellulase, although it hydrolyzes Avicel relatively easier than CM-cellulose. We obtained no evidence that this cellulase is an exo-type one named cellobiohydrolase (CBH) in the systematic nomenclature.

Flax Seed Proteins Comparison by Various PAGE‐Techniques in Slabs

AbstractSeed proteins of a number of Egyptian, French, and British cultivars of flax have been separately extracted with buffer, and water and buffer respectively, and analyzed by slab electrophoresis: PAGE, SDS‐PAGE, and poro‐SDS‐PAGE (22.5%–10%), and by PAGIF. The protein patterns were compared to find out the best method to differentiate between flax cultivars.The results of the water extracts and the residue extracted with Tris/borate buffer and analyzed on SDS‐PAGE indicated that the approximate MWs of native albumins and globulins ranged from 4.2 to 1.2 and 7.6 to 1.6 × 104, respectively. It is also shown that SDS‐PAGE of globulins extracted with Tris/borate buffer are the method recommended for differentiation of flax cultivars.The data of the total proteins extracted with Tris/HCl buffer and analyzed on SDS‐PAGE under reducing and non‐reducing conditions exhibited 5 disulphide bonded bands which on reduction gave acidic subunits and basic subunits.

Study of acid DNase in cow snout epidermis by micro slab electrophoresis using acidic and basic gel sheet.

Study of acid DNase in cow snout epidermis by micro slab electrophoresis using acidic and basic gel sheet.

Field inversion gel electrophoresis (FIGE) in vertical slabs as an improved method for large DNA separation.

Field inversion gel electrophoresis (FIGE) in vertical slabs as an improved method for large DNA separation.

Design of a vertical gel slab electrophoresis apparatus containing a permanent salt bridge

AbstractAn electrophoresis apparatus of the vertical slab gel type was designed to obtain a unit in which the number of spare parts and manual operations necessary for its assembly is significantly reduced as compared to the designs that are commonly used at present. The new apparatus is assembled in less than 60 s, and the gels are moulded in situ., i. e. without the use of a separate casting stand. With all electrode reservoirs placed within the base unit, the apparatus obviates the need for bulky external electrode reservoirs. The compact design is made possible by use of a permanently mounted porous plastic bridge that acts as a seal during moulding of the gel and as a conducting salt bridge during electrophoresis. The apparatus permits simultaneous running of two gels, and runs on single gels may be performed independently without filling the electrode reservoirs for the other gel.

Isolation of specific proteins affected by estradiol in the arcuate-median eminence of prepuberal female rats

Prepuberal female rats (25 days of age) were injected with estradiol benzoate (EB 10 μg/rat, s.c. in oil) or oil vehicle. Forty-eight hours after treatment, all animals were decapitated, their brains removed and sectioned. The arcuate nucleus of the hypothalamus and median eminence were microdissected and processed for isoelectric focusing followed by slab electrophoresis. The resulting two-dimensional electrophoretic gels were analyzed to quantitate the specific proteins resolved using a scanning microdensitometric method. Out of 235 proteins measured, 8 proteins were found to be significantly increased and 4 were decreased by EB treatment. The proteins which increased in concentration ranged in molecular weight from 15 to 43 kDa and isoelectric points (pI) of 4.9 to 7.0. The 4 proteins decreased by the EB treatment were 44, 67, 74 and 80 kDa in molecular weight and their pI's ranged from 6.5 to 7.1. It is suggested that these proteins might be involved in some of the neuroendocrine effects that are induced by estradiol in this region of the brain.