Recombinant peptides as immunogens: a comparison of protocols for antisera production using the pGEX system.

Using an inducible vector system that directs high-level production and rapid purification of recombinant protein, we have immunized mice with peptides prepared by several methods: 1) samples fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) subsequently transferred to PVDF membrane and subcutaneously implanted in mice; 2) samples cut directly from SDS-PAGE gels and injected intraperitoneally; 3) injection of recombinant protein bound to agarose beads; and 4) injection of log-phase E. coli transformed with recombinant vector. All four strategies yielded specific antisera reacting with both the parental fusion protein and the recombinant fragment as determined by enzyme-linked immunosorbent assay and immunoblot analysis. Specific recognition of the recombinant fragment was demonstrated by a competitive inhibition assay in which the parental fusion protein abrogated reactivity of serum with the isolated recombinant fragment.     

Enhanced sensitivity RNA gel loading buffer that enables efficient RNA separation on native gels

RNA gel analysis is essential for quality assessment of RNA preparations for subsequent analysis such as microarrays and real-time PCRs. The routinely used standard electrophoresis of RNA through formaldehyde-containing agarose gels is not only labor-intensive and time-consuming, but also involves sizeable quantities of hazardous materials. Above all, it is not sensitive, requiring more than 1 microgram of RNA for the assay. Current gene expression profiling with microarrays and real-time PCR often involves limiting amounts of RNA. It is therefore important to have a more sensitive way to analyze RNA. Here we report an improved ethidium bromide-based RNA gel analysis system with our Superload buffer that increases sensitivity to 12.5 ng of total RNA and allows RNA analysis on a regular native Tris-acetate EDTA (TAE) agarose gel.     

Adsorptive purification of pDNA on superporous rigid cross-linked cellulose matrix

Use of plasmid DNA (pDNA) in the emerging gene therapy requires pure DNA in large quantities requiring production of safe DNA on large scale. While a number of kit-based DNA purification techniques have become popular, large scale cost effective purification of DNA remains a technological challenge. Most traditional, as well as newly developed methods for DNA purification are expensive, tedious, use toxic reagents, and/or generally not amenable for scaled up production. Our attempts to develop a scalable adsorptive separation technology resulted in successful use of indigenously developed rigid cross-linked cellulose beads for single step purification of pDNA from alkaline cell lysates. This mode of purification employs a combination of intra-particle interactions that could give a product plasmid DNA free from chromosomal DNA, RNA and host proteins in a single scalable chromatographic step. The technology can be employed as a batch adsorption step on small scale, or on a large scale column chromatography. A high copy number 9.8 kb plasmid (from an Escherichia coli strain) was purified in yields of 77 and 52%, respectively in batch and column modes. The product obtained was homogeneous supercoiled plasmid with no RNA and protein contamination confirmed by quantitative analysis, agarose gel electrophoresis and SDS-PAGE.     

Purification of creatine kinase from beef heart mitochondria]

53-fold purified creatine kinase is isolated from beef heart mitochondria by phosphate buffer extraction followed by chromatography on DEAE-cellulose and KM-cellulose and preparative electrophoresis in phosphate buffer density gradient. The purified enzyme was homogenous under electrophoresis in agarose gel and moved to cathode. The enzyme did not enter into separating gel under disc electrophoresis in conditions for the separation of neutral anc acid proteins, while under conditions for separating alkaline proteins it produced five fractions. The stability of creatine kinase under storage considerably decreased after the purification.     

大腹园蛛鞭毛样丝蛋白cDNA克隆

应用RT-PCR技术,从大腹圆蛛（Araneus ventricosus)壶腹腺中扩增出鞭毛样丝蛋白基因（flagelldid-form silk protein gene),经1.5%琼脂糖凝胶电泳分离,WizardPCR Preps DNA Purification System回收后,将其克隆在pGEM-T载体中,经限制性核酸内切酶鉴定和核苷酸序列分析证实,构建的重组擀粒pSF1中含有蜂蛛鞭毛样丝蛋白基因,且含有3个重复序列。     

Automated one-step DNA sequencing based on nanoliter reaction volumes and capillary electrophoresis

An integrated system with a nano-reactor for cycle-sequencing reaction coupled to on-line purification and capillary gel electrophoresis has been demonstrated. Fifty nanoliters of reagent solution, which includes dye-labeled terminators, polymerase, BSA and template, was aspirated and mixed with the template inside the nano-reactor followed by cycle-sequencing reaction. The reaction products were then purified by a size-exclusion chromatographic column operated at 50°C followed by room temperature on-line injection of the DNA fragments into a capillary for gel electrophoresis. Over 450 bases of DNA can be separated and identified. As little as 25 nl reagent solution can be used for the cycle-sequencing reaction with a slightly shorter read length. Significant savings on reagent cost is achieved because the remaining stock solution can be reused without contamination. The steps of cycle sequencing, on-line purification, injection, DNA separation, capillary regeneration, gel-filling and fluidic manipulation were performed with complete automation. This system can be readily multiplexed for high-throughput DNA sequencing or PCR analysis directly from templates or even biological materials.     

Recycling agarose

The cost of agarose for DNA gel electrophoresis can be significant in largescale projects. The International Potato Center (CIP) laboratory in Lima-Perú reuses agarose to save money. Due to limited financial resources for research, not uncommon in Latin American laboratories, this practice satisfies a need to control expenses. Recycled agarose can be used for RAPD analysis, or other applications in which no further manipulation of the DNA is necessary. Here we describe an improved CIP method where large amounts of agarose are ultimately equilibrated in water and dried to a powder similar to the original agarose.     

Restriction fragment length polymorphism analysis of ERBB2 oncogene by capillary electrophoresis

Capillary electrophoresis (CE) with a sieving buffer containing ethidium bromide was applied to the detection of PCR-amplified RFLP samples. With CE, in contrast to agarose gel electrophoresis, run times are short, i.e., typically less than 30 min, the capillary can be re-used, and full automation is feasible. The addition of ethidium bromide to the buffer system in conjunction with a field amplification injection technique led to increased sample detectability and resolution. Migration time precision was better than 0.2% RSD with a approximately 12-bp resolution for the DNA fragment sizes of interest. RFLP samples were analyzed for homo- or heterozygosity based on the presence of 500- and/or 520-bp DNA fragments. Special software was used to correct for run-to-run migration time variations, thus facilitating genotype assignment.     

Purification of Uricase by Biospecific Adsorption-Desorption

A simple procedure for the purification of uricase from bovine kidney is described. The procedure involves the following steps: 1) processing of kidney mince by borate/butanol, 2) ammonium sulphate precipitation, and 3) biospecific adsorption-desorption. The adsorbents were prepared by chemical attachment of urate or xanthine to agarose gel beads. The desorption was performed by a xanthine solution. The adsorption-desorption procedure resulted in an 11 000–12 000-fold purification. The specific activity of the purified uricase was 19.8 U/mg using either “urate” or “xanthine” adsorbent. The recovery was about 70%.The adsorbents were also used for the purification of commercial uricase preparations from hog liver. In this case the purified uricase also possessed a specific activity of 19.8 U/mg. The products were homogenous as judged by gradipore electrophoresis and gel filtration.     

Purification of uricase by biospecific adsorption-desorption

A simple procedure for the purification of uricase from bovine kidney is described. The procedure involves the following steps: 1) processing of kidney mince by borate/butanol, 2) ammonium sulphate precipitation, and 3) biospecific adsorption-desorption. The adsorbents were prepared by chemical attachment of urate or xanthine to agarose gel beads. The desorption was performed by a xanthine solution. The adsorption-desorption procedure resulted in an 11 000-12 000-fold purification. The specific activity of the purified uricase was 19.8 U/mg using either "urate" adsorbent. The recovery was about 70%. The adsorbents were also used for the purification of commercial uricase preparations from hog liver. In this case the purified uricase also possessed a specific activity of 19.8 U/mg. The products were homogenous as judged by gradipore electrophoresis and gel filtration.     

High-density small-volume gel loading directly from capillary tubes

A technique has been developed for high lane density loading of small-volume DNA samples in a horizontal agarose gel. This technique has been investigated with a simple hand-held tool that is made to couple to sample output from a new capillary-based sample automation system. The approach consists of piercing the gel with pressurized sample capillaries and relieving the pressure shortly before withdrawal. The pressurization prevents the capillary from aspirating the gel buffer and keeps the sample at the tip of the capillary, so that it may be sucked into the gel during withdrawal. This method is shown to be adequate for a wide range of DNA ladders and PCR-based screening. In addition to allowing smaller lanes and a higher lane density than is achievable with traditional well-forming techniques, it relaxes the need for well formation and the alignment of the sample loader with those wells, providing an easy, efficient means of loading agarose gels.     

Simultaneous use of standard and low-melting agarose for the separation and isolation of DNA by electrophoresis

In this communication we describe the sequential use of standard and low-melting agarose in a single gel slab for the electrophoresis of DNA. This method has the advantages of high resolution and reproducibility characteristic of standard agarose and the ease of manipulation of DNA for direct cloning, sequential digestion and isolation, characteristic of low-melting agarose.     

Two-dimensional agarose gel electrophoresis without gel manipulation

The apparatus and procedure to perform two-dimensional agarose gel electrophoresis without manipulating the gel used for the first electrophoresis (first-dimension gel) have been developed. The procedure is less complex, less damaging to first-dimension gels, and more precise than procedures that require manipulation of the first-dimension gel. When combined with gel-embedding techniques, the procedure presented can be used to perform the second electrophoresis in a gel different from the first-dimension gel. A first-dimension gel too dilute to be manipulated and a more concentrated gel for the second electrophoresis have been used to separate DNA open circles from a mixture of variable-length linear DNAs.     

Agarose gel electrophoresis of denatured RNA with silver staining

This paper describes agarose gel electrophoresis and silver staining of denatured RNAs. Glyoxal- or formaldehyde-denatured RNAs are electrophoresed in an agarose gel cast on a plastic support using an inert, low conductivity buffer. Following electrophoresis, the gel is stained with a sensitive silver stain. The method produces sharp, well-resolved bands and yields accurate RNA size estimates. Because of its sensitivity and simplicity, it is suitable for routine laboratory use.     

A fast and convenient MALDI-MS based proteomic approach: identification of components scaffolded by the actin cytoskeleton of activated human thrombocytes

A recently developed concentration and purification method (Gevaert, K., Demol, H., Puype, M., Broekaert, D., De Boeck, S., Houthaeve, T., Vandekerckhove, J., 1997. Electrophoresis 18, 2950-2960) for the analysis of diluted peptide samples by matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) is compared with conventional MALDI sample preparation methods. In the procedure developed, reverse-phase chromatographic beads are added to diluted peptide solutions and act as a peptide-trapping device. Peptides concentrated on the added beads are subsequently harvested, transferred to the MALDI-target disc and efficiently on target desorbed from the beads in a very small volume of an organic-aqueous mixture containing the aromatic MALDI-matrix components. Using this procedure, we show that it is possible to use the totality of in gel protein digests without negative interference of buffers and chaotropes that may be present in the digestion mixture. This method links MALDI-MS peptide analysis more efficiently to 2-D gel electrophoresis in the concept of proteome analysis. The procedure is illustrated by the identification of a class of proteins, which translocate to the actin cytoskeleton of human platelets upon thrombin stimulation.     

Mapping genomic organization by field inversion and two-dimensional gel electrophoresis: application to the murine T-cell receptor gamma gene family.

A new two-dimensional gel electrophoresis technique has been developed for the mapping of multigene families. Resolution in the first dimension is based on the generation of large size DNA fragments by infrequently-cutting restriction enzymes, and separation of these fragments by field inversion gel (FIG) electrophoresis. A second restriction enzyme digestion is then carried out with the separated DNA fragments in the agarose gel. Standard gel electrophoresis in the second dimension allows one to estimate the number of hybridizing genes contained in each large DNA fragment. We have also developed a novel method to increase the separation, resolution and hybridization signal in the second dimension by condensing the bands from the first dimension into spots. As an example, we have applied these techniques to determine the organization of the murine T-cell receptor gamma locus. The murine gamma gene family was found to be contained on two DNA fragments encompassing 195 kilobases of DNA. The two-dimensional gel electrophoresis method is particularly useful in the analysis of the organization of multigenic families where single copy probes are not readily available, and should extend the potential usefulness of field inversion gel electrophoresis in gene mapping.     

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.     

Purification and some of the functions of the products of bacteriophage T4 recombination genes, uvsX and uvsY

The nonessential T4 genes uvsX and uvsY are involved in DNA repair and general recombination. Using newly isolated amber mutants of these genes, we have identified the gene products (gp) by sodium dodecyl sulfate (SDS)/polyacrylamide gel electrophoresis. Their relative molecular masses are 39 000 and 16 000, respectively. In the normal wild-type infection process they are produced early but not late in infection. Their synthesis continues for a longer period when DNA synthesis is blocked. We have developed procedures to isolate these gene products at a purity of more than 95% for gpuvsX and at 70% for gpuvsY, as judged by SDS/polyacrylamide gel electrophoresis and staining with Coomassie brilliant blue dye. The purification procedures suggest that these products may be membrane proteins. Using both an agarose gel assay and electron microscopy, we find that the product of the gene uvsX catalyzes the assimilation of a linear single-stranded fd DNA fragment into superhelical double-stranded fd DNA (RFI). The reaction requires ATP and Mg2+ besides substrate DNAs and uvsX protein. The T4 uvsX protein therefore is similar to the Escherichia coli recA protein in molecular size and function, but differs in antigenic property.     

Application of fluorescence-based resistance gene analog analysis for genotyping plant genetic resources

Conventional approaches for detecting disease resistance gene analogs (RGAs) in plants are based on agarose gels or on polyacrylamide gel electrophoresis (PAGE) in combination with silver staining or radioactive labeling. A modified method for RGA analysis has been developed by using fluorescence-labeled primers for PCR amplifications. The amplified fragments are detected by denaturing PAGE using an automated laser fluorescence DNA sequencer and analyzed by fragment analysis software. This technique is not limited to specific plant species and is suitable for high-throughput genotyping plant genetic resources. We demonstrate here the efficiency of this method for comparison of RGA patterns in diverse plant species and for genotyping of natural populations of the wheat progenitor, Triticum dicoccoides. Revisions requested 8 October 2004; Revisions received 15 November 2004     

Purification of muscarinic acetylcholine receptors by affinity chromatography

Calf forebrain homogenates contain 2.8 pM muscarinic acetylcholine receptors per mg of protein. [3H]Antagonist saturation binding experiments under equilibrium conditions revealed a single class of sites with equilibrium dissociation constants of 0.82 nM for [3H]dexetimide and 0.095 nM for [3H]quinuclidinyl benzilate. Displacement binding studies with agonists revealed the presence of low and high affinity sites. Here we describe the solubilization of muscarinic acetylcholine receptors with digitonin and their purification by affinity chromatography using an affinity gel which consisted of dexetimide coupled to Affi-Gel 10 (i.e., carboxy N-hydroxysuccinimide esters linked via a 1 nm spacer arm to agarose beads). Purified proteins were obtained by specific elution with muscarinic drugs, i.e., the antagonist atropine and the irreversible ligand propylbenzilylcholine mustard. SDS-polyacrylamide gel electrophoresis of the radioiodinated purified preparations revealed a major 70-K protein.