Isoelectrical focusing of connectin by agarose gel electrophoresis

Two-dimensional gel electrophoresis using 0.5% agarose gel in the 1st dimension and 2-12% gradient polyacrylamide gel in the 2nd dimension succeeded in the isoelectrical focusing of connectin, a giant myofibrillar protein of approximately 3,000 kDa. Immunoblotting with an anti-connectin monoclonal antibody, SM1-36-2, following the two-dimensional gel electrophoresis, demonstrated that connectin was an acidic protein with an estimated pI of approximately 5.7.     

Purification of coated vesicles by agarose gel electrophoresis

We have applied agarose gel electrophoresis as a novel step in the purification of clathrin-coated vesicles. Preparations of coated vesicles obtained by sedimentation velocity and isopycnic centrifugation are resolved into two distinct fractions upon electrophoresis. The slower migrating fraction contains smooth vesicles, whereas the faster contains only coated vesicles and empty clathrin coats. The faster mobility of the coated vesicles is primarily caused by the acidic nature of clathrin. Coated vesicles from three different cell types have different mobilities. In each case, however, all of the major polypeptides previously attributed to coated vesicles comigrate with the now homogeneous particles, even though a powerful ATPase activity is completely removed.     

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.     

Sizing of the Leptospira genome by pulsed-field agarose gel electrophoresis

Pulsed-field gel electrophoresis allowed the determination of the size of the genome of Leptospira, a bacterium of the spirochete family. The three restriction enzymes, NotI (5'GC/GGCCGC), NheI (5'G/CTAGC), ApaI (5'-GGGCC/C) generated DNA fragments of suitable size. The results are compatible with a size of 5000 kb for the chromosome of both the pathogenic and the saprophytic species of Leptospira.     

Characterization of eight VNTR loci by agarose gel electrophoresis

Allelic frequencies and their confidence intervals were obtained for eight independent VNTR loci from a sample of more than 75 Utah Caucasians. Using high-resolution agarose gel electrophoresis, we were able to resolve alleles at the D17S5 locus that differed by only one repeating unit; it was therefore possible to name the alleles according to the number of repeating units each contained. Two a priori probabilities were calculated for each VNTR locus separately and for all eight loci jointly: (i) the "power of exclusion" for an alleged father/mother/child trio and for an alleged parent/child duo, and (ii) the "probability of matching" when two unrelated individuals or two siblings are genotyped.     

一种改进的甲醛—琼脂糖凝胶电泳

构建ｃＤＮＡ文库,进行Ｎｏｒｔｈｅｒｎ杂交分析以及ＲＴ－ＰＣＲ等分子生物学研究,都需要纯度高、完整性好的ＲＮＡ。鉴定ＲＮＡ的完整性通常需要进行甲醛－琼脂糖凝胶电泳。然而,传统的甲醛－琼脂糖凝胶电泳操作步骤繁杂［１］,本文提出一种改进的甲醛－琼脂糖凝胶电泳,不仅简化了操作,而且效果很好。我们以向日葵总ＲＮＡ和ｃＤＮＡ甲醛－琼脂糖凝胶电泳为例,对这一方法进行介绍。１ 材料与方法１．１ 总ＲＮＡ的提取及ｍＲＮＡ的分离：取向日葵花蕾１ｇ,按照改进的异硫氰酸胍法提取向日葵总ＲＮＡ［２］。利用磁珠法分离ｍＲ－…     

An inexpensive agarose gel electrophoresis system

This article describes the design and construction of an inexpensive apparatus for agarose gel electrophoresis. The gel unit comprises an empty pipette tip box fitted with aluminum foil electrodes. A schematic is also provided for a DC power supply composed of simply a transformer and diodes. Photographic evidence is presented to document the resolution and utility of these devices.     

Population genetics of red cell galactose-1-phosphate-uridyl-transferase (EC: 2.7.7.12)

Summary The genetic polymorphism of galactose-1-phosphate-uridyl transferases was investigated in a population sample in southwestern Germany. The frequency of the common allele Gt² was estimated to be 0.0723. Due to the electrophoretic pattern of homozygous and heterozygous phenotypes the enzyme is of dimeric structure consisting of two identical polypeptide chains.

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Zusammenfassung Der Polymorphismus der Galactose-1-Phosphat-Uridyl-Transferasen wurde in einer Bevölkerungsstichprobe aus Südwestdeutschland untersucht. Die Genfrequenz für das Allel Gt² beträgt 0,0723. Aus den Zymogrammen ergibt sich, daß das Enzyme Dimerstruktur besitzt.

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Supported by the Deutsche Forschungsgemeinschaft.     

Unidirectional replication as visualized by two-dimensional agarose gel electrophoresis

Two-dimensional (2D) agarose gel electrophoresis is progressively replacing electron microscopy as the technique of choice to map the initiation and termination sites for DNA replication. Two different versions were originally developed to analyze the replication of the yeast 2 microns plasmid. Neutral/Neutral (N/N) 2D agarose gel electrophoresis has subsequently been used to study the replication of other eukaryotic plasmids, viruses and chromosomal DNAs. In some cases, however, the results do not conform to the expected 2D gel patterns. In order to better understand this technique, we employed it to study the replication of the colE1-like plasmid, pBR322. This was the first time replicative intermediates from a unidirectionally replicated plasmid have been analyzed by means of N/N 2D agarose gel electrophoresis. The patterns obtained were significantly different from those obtained in the case of bidirectional replication. We showed that identification of a complete are corresponding to molecules containing an internal bubble is not sufficient to distinguish a symmetrically located bidirectional origin from an asymmetrically located unidirectional origin. We also showed that unidirectionally replicated fragments containing a stalled fork can produce a pattern with an inflection point. Finally, replication appeared to initiate at only some of the potential origins in each multimer of pBR322 DNA.     

Restriction analysis of the Streptomyces glaucescens genome by agarose gel electrophoresis

A method for the analysis of total DNA of Streptomyces glaucescens is described. The relevant steps are (a) extraction and purification of DNA, (b) restriction of DNA samples with type II restriction enzymes, (c) one dimensional separation of restriction fragments by agarose gel electrophoresis. A typical banding pattern was obtained for each wild type strain, independant of growth conditions or age of the culture. Mutant strains exhibited in most cases the same banding pattern as the parent wild type strain. Only in one specific mutant class a fragment of about 9 megadalton was missing.     

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.     

Determination of a particle's radius by two-dimensional agarose gel electrophoresis

Electrophoresis in an agarose gel dilute enough to be almost nonretarding, followed by electrophoresis in an orthogonal direction into a more concentrated agarose gel, has been developed as a procedure to determine the radius of spherical particles. Unlike procedures of unidirectional electrophoresis in a single gel, the above procedure can be used to compare the radii of particles that differ in solid-support-free electrophoretic mobility. Accuracy of 0.3 nm has been achieved with particles 30 nm in radius. It was found that the apparent radius of the spherical capsid of bacteriophage P22 decreased by 3% during elevated temperature-induced ejection of DNA from the capsid. Though originally designed for use with multimolecular particles, the procedure described here should also be useful with monomolecular particles.     

Analysis of guanase by agarose gel electrophoresis and activity staining

A method was developed to separate guanase by agarose gel electrophoresis and to detect its activity by staining of the bands with a mixture of the enzymes xanthine oxidase, catalase, and aldehyde dehydrogenase, the coenzyme NADP+, and a substrate of guanine, ethanol, phenazine methosulfate, nitrotetrazolium blue, and KCN in Tris-(hydroxymethyl)methylamine buffer (pH 8.0). Serum samples showed bands 1 (faster moving) and 2 corresponding to the positions of albumin and alpha 2-globulin, respectively, found by serum protein staining. The same bands were detected with guanase from human liver and kidney, although band 2 from the latter samples was not as distinct as that from the liver samples.