Agarose-acrylamide gradient gel electrophoresis of proteins

A new agarose-acrylamide gradient slab gel electrophoresis system is described. The preparation of this new gel has been facilitated by the use of agarose with a relatively low gelation temperature. Fractionation of marker proteins and crosslinked proteins from a subcellular cytoskeletal preparation on agarose-acrylamide gradient gels is compared to that found using other acrylamide gel electrophoresis systems.     

Behavior of rat liver catalase during electrophoresis in a pH gradient.

Investigations were conducted on the distribution of rat liver catalase subsequent to electrofocusing in a pH gradient. Differences were observed depending on the enzyme being extracted from the total mitochondrial fraction, from the supernatant of the homogenate or from purified peroxisomes. Catalase solubilized from the total mitochondrial fraction exhibits an apparent isoelectric point lower than that of catalase derived from the supernatant. Catalase released from purified peroxisomes shows a behavior similar to that of the supernatant catalase. It has been concluded that, in a total mitochondrial fraction, a factor is present that alters the electric charge of the catalase molecule during or after the extraction of the enzyme. This factor is probably associated with lysosomes existing together with peroxisomes and mitochondria in a total mitochondrial fraction. As a matter of fact, the addition of an extract of purified lysosomes to purified peroxisomes or to supernatant will cause a shift towards a more acid pH of catalase distribution subsequent to electrofocalization.     

Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology

Here, the state of the art of the application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology will be presented. Furthermore, the potentials and limitations of these techniques will be discussed, and it will be indicated why their use in ecological studies has become so important.     

Mobility measurements by photometric analysis of zone electrophoresis in a sucrose gradient column

A density gradient zone electrophoresis apparatus has been designed for measuring electrophoretic mobilities on small amounts (about 10 μg) of viruses or other ultraviolet-absorbing materials. Dialysis membranes separate the gradient column from electrode chambers and eliminate the need to maintain hydrostatic equilibrium between the buffer chambers. The virus zone was located by pumping the gradient column through an ultraviolet flow cell, a procedure which did not disturb the virus zone. The apparatus was tested by measuring the mobilities of tobacco mosaic and brome mosaic viruses in different buffers, in sucrose, glucose, and glycerol gradients, at 0° and 15°, and during ascending and descending electrophoresis.     

SDS microslab linear gradient polyacrylamide gel electrophoresis

An improved sodium dodecyl sulfate (SDS) microslab linear gradient polyacrylamide gel electrophoresis (PAGE) technique has been developed. Several important features present in this microslab SDS-PAGE system include (1) high resolution and sensitivity; (2) rapid electrophoresis, staining, and destaining; (3) high reproducibility; and (4) low cost of construction and operation. Several gels are east at once between unmodified commercially available microslides separated by 0.5-mm thick Teflon spacers. The total time from start of electrophoresis to completion of destaining spans 2 hr. Gels are dried between transparent cellophane membranes in 1 hr and can be easily scanned with a microdensitometer. As little as 20 ng of a purified protein stained with Coomassie blue is detectable.     

Dinoflagellate community analysis of a fish kill using denaturing gradient gel electrophoresis

A molecular method using the polymerase chain reaction (PCR) amplification of small subunit gene sequences (18S rDNA) and denaturing gradient gel electrophoresis (DGGE) was used to determine both the population complexity and species identification of organisms in harmful algal blooms. Eighteen laboratory cultures of dinoflagellates, including Akashiwo, Gymnodinium, Heterocapsa, Karenia, Karlodinium, Pfiesteria, and Pfiesteria-like species were analyzed using dinoflagellate-specific oligonucleotide primers and DGGE. The method is sensitive and able to determine the number of species in a sample, as well as the taxonomic identity of each species, and is particularly useful in detecting differences between species of the same genus, as well as differences between morphologically similar species. Using this method, each of eight Pfiesteria-like species was verified as being clonal isolates of Pfiesteria piscicida. The sensitivity of dinoflagellate DGGE is approximately 1000 cells/ml, which is 100-fold less sensitive than real-time PCR. However, the advantage of DGGE lies in its ability to analyze dinoflagellate community structure without needing to know what is there, while real-time PCR provides much higher sensitivity and detection levels, if probes exist for the species of interest, attributes that complement DGGE analysis. In a blinded test, dinoflagellate DGGE was used to analyze two environmental fish kill samples whose species composition had been previously determined by other analyses. DGGE correctly identified the dominant species in these samples as Karlodinium micrum and Heterocapsa rotundata, proving the efficacy of this method on environmental samples. Toxin analysis of a clonal isolate obtained from the fish kill samples confirmed the presence of KmTx2, corroborating the earlier genetic identification of toxic K. micrum in the fish kill water sample.     

Separation of lipooligosaccharides by linear gradient gel electrophoresis

Lipooligosaccharides (LOSs) are one of the major antigenic and immunogenic components on the outer membrane of mucosal Gram-negative bacteria. These glycolipid antigens are in the M(r) range of 3-7 kDa, and SDS/PAGE has been used as an analytical tool. Although we are able to separate relatively higher M(r) LOS components by mini-PAGE, we encounter difficulties in resolving LOS components below 3.6 kDa present in heterogeneous LOS preparations. In the present study, we selected PID2 LOS consisting of six LOS components of 3.0-5.1 kDa as a model LOS and examined mini-PAGE conditions not only to resolve smaller M(r) LOS components but also to retain resolving capability of higher LOS components. We found that mini-PAGE with stepwise and linear gradient gels (glycine-SDS) resolved smaller M(r) LOS components. Mini-PAGE with linear gradient gels gave the best resolution, and LOS components of 3.0-5.1 kDa were separated as tight and even bands. Because of the resolution, LOS components were stained chemically and immunochemically much better than those on continuous or stepwise gradient gels. Our study also showed that preformed tricine-SDS (TSDS) minigels such as 16.5 and 10-20% (linear gradient) did not resolve PID2 LOS, which indicated that heterogeneous LOS preparations may not be fully analyzed by using these TSDS minigels. By using glycine-SDS linear gradient mini-PAGE, we should be able not only to screen expression of LOSs but also to characterize smaller M(r) LOS components present in heterogeneous LOS preparations whose identities may have been neglected in the past.     

变性梯度凝胶电泳装置及其在DNA突变检测中的初步应用

设计了一套适用于变性梯度凝胶电泳(DGGE)的装置。该装置为带缓冲液循环系统、可精确控制温度的夹芯式双垂直电泳槽,其整个凝胶板内的控温精度和均匀度均在±0.5℃范围内。对其在DNA突变检测中的应用作了初步的探讨。通过PCR-DGGE连用成功地分离了野生型p53基因和含点突变的P53基因(第141密码子由TGC突变为TAC)。     

Characterization of benthic bacterial assemblages in a polluted stream using denaturing gradient gel electrophoresis

To study differences in bacterial assemblages among sites with different environmental conditions, sediment samples were collected from three sites along a South Carolina (U.S.A.) coastalplain stream with varying levels of anthropogenic perturbation. The objective of this study was to compare the bacterial assemblages among these sites to detect possible impacts from the disturbance. To accomplish this comparison, DNA was extracted from the samples and subjected to the polymerase chain reaction using primers designed to amplify bacterial 16S rRNA genes. Relative measures of bacterial genetic diversity, assessed using denaturing gradient gel electrophoresis (DGGE), revealed greater numbers of unique sequences at the disturbed sites. The number of bands, which is analogous to species richness, did not vary predictably among sites. Similarity indices revealed a high level of similarity among replicate samples from each site and low similarity between samples from different sites. This study demonstrated that bacterial assemblages differed among sites and that the presence or absence of certain species, represented by unique DGGE bands, differed among sites; unique bands were most commonly encountered at the disturbed sites. Based on the evidence gathered, we conclude that benthic bacterial assemblages vary longitudinally and that anthropogenic disturbance may alter the bacterial component of streams.     

变性梯度凝胶电泳技术在微生态学研究中应用的进展

变性梯度凝胶电泳(Denaturing gradient gel electrophoresis,DGGE)技术具有无需进行微生物培养、检测率高、分辨率高、重复性好等优点,现已逐渐成为微生态学研究中的强有力工具.本研究介绍了变性梯度凝胶电泳(DGGE)的基本原理和实验步骤中的系统优化,重点介绍和讨论了DGGE在微生态学研究中应用以及进展情况,并对该技术的应用前景进行了展望.