Electrophoretically silent alleles in a finite population

Summary The expected number of silent alleles in an electromorph is computed for various values of population size (N), mutation rate (u), and sample size (s) under the assumption of no selection. The proportion of alleles undetectable by electrophoresis is higher when Nu is large than when this is small. It is shown that an electromorph of high population frequency has more silent alleles than an electromorph of low frequency if the sample size is the same.     

Enhanced protein recovery after electrotransfer using square wave alternating voltage

Protein identification is becoming a complement to the available fully sequenced genomes. To meet the challenge, newly developed techniques for high throughput protein identification using matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) and peptide mass fingerprint are needed. Two years ago, a parallel protein digestion process was proposed. It provided a collecting polyvinylidene difluoride (PVDF) membrane able to be scanned by MALDI. Acquired data were used to recreate a virtual multidimensional image. Voltage used during this protein electroblotting technique was an unusual square wave alternative voltage (SWAV). The goal of the current study is to evaluate quantitatively the efficiency of the SWAV compared with a classical electroblot process on intact proteins. The effect of the pulsed electric field and the buffer composition were compared to a standard continuous transblotting process defined as the gold standard. Combination of the pulsed asymmetric electric field with 3-(cyclohexylamino)-1-propane-sulfonique acid (CAPS) buffers showed an average 65% increase of protein recovery. Moreover, a strongest effect is observed for high M(r) proteins. In conclusion, the present study highlighted a positive influence of the "shaking" effect of the asymmetric alternative voltage on gel protein extraction.     

Utility of a direct dual-mode development analysis on blotted protein mixtures

Classic blotting methods remain a commonly applied approach to specific protein identification in gel electrophoresis of complex mixtures despite the inherent difficulty in band or spot matching due to significant variability of protein migration or localization in replicate blotting experiments. A direct application of both protein stain and protein blotting on a single membrane significantly reduces the complexity of the experiment and provides increased confidence of signal matching. Digital alignment of images acquired from both total protein stain and blotting development modes on a single membrane allows unambiguous spot or band assignments in these experiments as well as retention of quantitative information acquired from both modes of signal generation. A direct and simple method applying a fluorescent protein stain that is compatible with subsequent detection by antibody or lectin recognition factors along with common image adjustment software is examined. The utility of this blot dual-mode development method for direct protein recognition and quantification in one- and two-dimensional electrophoresis is demonstrated for bioanalytical objectives where replicate experiments are challenged by sample complexity.     

Schistosoma japonicum: disc electrophoretic protein patterns of the Japanese, Philippine, and Formosan strains

Soluble proteins of the Japanese, Philippine, and Formosan strains of Schistosoma japonicum were separated by disc electrophoresis using polyacrylamide gel columns. Differences between strains and sexes were investigated on the basis of R_f values of the bands, location of prominent peaks, quantitative comparisons of major bands, and overall densitometric profiles. With male extract, 29, 28, and 29, distinct bands were resolved for the Japanese, Philippine, and Formosan strains, respectively. Female extracts gave 31, 26, and 25 distinct bands for the Japanese, Philippine, and Formosan strains, respectively. Both qualitative and quantitative differences were found among the three strains and between sexes. The closest relationship of densitometric profile was between the Japanese and Formosan strains.     

The detection of genetic variation in Leptospira interrogans serogroup ICTEROHAMORRHAGIAE by ribosomal RNA gene restriction fragment patterns

Twenty-eight isolates of catalase-negative/weak (CNW) thermophilic campylobacters from human blood and faecal cultures were characterized by one-dimensional (1-D) high-resolution SDS-PAGE of cellular proteins. A further 11 Campylobacter strains were included for reference purposes. Partial protein patterns were used as the basis for numerical analysis, which showed that all of the hippurate-positive strains had a high similarity to C. jejuni. Two subclusters were formed within C. jejuni corresponding to C. jejuni subsp. doylei (15 strains) and C. jejuni subsp. jejuni (4 strains). Most of the paediatric strains from South Africa were members of C. jejuni subsp. doylei. Hippurate-negative CNW thermophilic strains were identified as "C. upsaliensis". The analysis demonstrated that the catalase-negative C. jejuni strains were quite distinct from "C. upsaliensis" and that electrophoretic protein patterns provide an excellent criterion for the identification of subspecies within C. jejuni.     

Protein Turnover in Retina

Abstract: Rabbit retinas were exposed in vitro to 0.5-h pulses of [³H]leucine or [¹⁴C]Ieucine. Some retinas were harvested promptly after labeling to measure synthesis. These were combined, in double-labeling experiments, with retinas that had been returned to unlabeled medium for a subsequent 1 h or 3.75 h to measure degradation. All of the proteins were solubilized, and separated according to size by gel electrophoresis. The gels were cut into 95 slices, and each slice was differentially counted. The amount of protein in the slice was estimated from the Coomassie blue staining, and its molecular weight from the distribution of molecular weight (MW) standards. Turnover rates of the various sizes of proteins were calculated from these data using certain well-defined assumptions. Retinal protein contained about 32 ± 10³ nmol of polypeptide per g, with a median MW of 27,000. Total synthesis was at the rate of 103 nmol/g of protein/h, with the most rapid synthesis in the 33,000–43,000 MW range, at 2 nmol/g/h for every 1000 increment in MW. Protein renewal averaged 0.52%/h, but varied directly (p < 0.0001) with MW, so that proteins of 10,000 MW were being renewed at about 0.1%/h and proteins of 140,000 MW at about 1.4%/h. Taken together, the measurements of fractional renewal and the measurements of degradation of the newly synthesized proteins demonstrated that each slice contained proteins with markedly different breakdown coefficients, and provided enough information to characterize the proteins in the slice in terms of a fast and a slow subgroup. This analysis indicated that: breakdown coefficients varied much more than rates of synthesis and were therefore the prime determinant of the amount of each protein that was present; as MW increased, breakdown coefficients of the long-lived proteins increased (p < 0.0001), accounting in major part for the correlation between size and turnover; most staining bands were due to proteins with peculiarly long lifespans; the proteins with the slowest turnover of all appeared to be histones: there was an unusually rapid synthesis of a 138,000 MW polypeptide with a moderately short half-life (about 3 h).     

花生种子老化相关蛋白质的初步研究

本文以粤油 116花生(Arachis hypogaea L.)为材料,对不同处理种子的除子叶“种胚”(以下简称“种胚”)的蛋白质进行了研究.实验结果表明,当花生种子活力下降到一定程度时,其“种胚”内出现一种新蛋白质( pI6.2、MW 10 KD),随种子老化程度加深,含量逐渐增多.我们认为该蛋白质与花生种子老化存在着一定的相关关系,可作为该种子老化的标志.     

花生种子老化相关蛋白质的初步研究

本文以粤油 116花生(Arachis hypogaea L.)为材料,对不同处理种子的除子叶“种胚”(以下简称“种胚”)的蛋白质进行了研究.实验结果表明,当花生种子活力下降到一定程度时,其“种胚”内出现一种新蛋白质( pI6.2、MW 10 KD),随种子老化程度加深,含量逐渐增多.我们认为该蛋白质与花生种子老化存在着一定的相关关系,可作为该种子老化的标志.     

Analysis of the Shewanella oneidensis proteome by two-dimensional gel electrophoresis under nondenaturing conditions

Proteomes are dynamic, i.e., the protein components of living cells change in response to various stimuli. Protein changes can involve shifts in the abundance of protein components, in the interactions of protein components, and in the activity of protein components. Two-dimensional gel electrophoresis (2-DE) coupled with peptide mass spectrometry is useful for the analysis of relative protein abundance, but the denaturing conditions of classical 2-DE do not allow analysis of protein interactions or protein function. We have developed a nondenaturing 2-DE method that allows analysis of protein interactions and protein functions, as demonstrated in our analysis of the cytosol and crude membrane fractions of the facultative anaerobe Shewanella oneidensis MR-1. Our experiments demonstrate that enzymatic activity is retained under the sample and protein separation methods described, as shown by positive malate dehydrogenase activity results. We have also found protein interactions within both the soluble and membrane fractions. The method described will be useful for the characterization of the functional proteomes of microbial systems.     

Lipopolysaccharide aggregates in native agarose gels detected by reversible negative staining with imidazole and zinc salts

We investigated the use of imidazole and zinc salts for the detection of lipopolysaccharide (LPS) aggregates separated by native agarose gel electrophoresis (NAGE). As a result, a new staining procedure was established by which as little as 1.5 μg of Escherichia coli O55:B5 LPS aggregates was detected by means of inducing a clear, transparent pattern, contrasted against an opaque background. E. coli O55:B5 LPS preparations treated with nucleases and proteinase K proved that the reverse-stained LPS pattern is not related to any potential artifacts caused by unrelated biomolecules (e.g., nucleic acids, proteins). After this, we showed that the procedure is applicable to two-dimensional LPS separation using NAGE/SDS-PAGE, while at the same time confirming that real polydisperse LPS aggregates are represented by the stained profile. Also, we demonstrated the general applicability of this stain to the detection of different NAGE-separated LPS aggregates (e.g., from E. coli 026:B6, E. coli 0111:B4, Salmonella minnesota Re595). Finally, using lysozyme as a model protein, we found that imidazole–zinc may be combined with Coomassie brilliant blue R-250 into a double-staining process to enable the use of NAGE for investigating the interaction of cationic proteins and LPS aggregates and protein or LPS concentration effects on protein–LPS binding.     

泡桐叶片蛋白质多态性及其聚类分析

研究了9种泡桐叶片蛋白质的多态性,并根据其叶片蛋白质聚丙烯酰胺凝胶单向和双向电泳结果,将它们聚类为白花泡桐组[白花泡桐（Paulownia fortunei）和白花兰泡桐（P.elongata f.allba）]、南方泡桐组[南方泡桐（P.australis )和成都泡桐（P.albiphloea var.chengtuensis)]和毛泡桐组（毛泡桐（P.tomentosa）、川泡桐（P.fargesii）、鄂川泡桐（P.albiphloea）、山明泡桐（P.lamprophylla）和兰考泡桐（P.elon-gata）]。该结果可为了解泡桐属植物的亲缘关系和种的鉴定提供参考依据。     

Improved method for simultaneous isolation of proteins and nucleic acids

Here we combine the use of fluorescence-enhancing silicon substrates coated by copoly(DMA–NAS–MAPS), a ter-copolymer based on N,N-dimethylacrylamide (DMA), N-acryloyloxysuccinimide (NAS), and 3-(trimethoxysilyl)propyl-methacrylate (MAPS), with an efficient dynamic incubation to overcome mass transport limitations and obtain femtomolar limits of detection. The high sensitivity was obtained with a conventional microarray scanner without the use of any sophisticated detection strategy or protocol. When the method was applied, an improvement of the analytical sensitivity of approximately three orders of magnitude was achieved for antibody detection when compared with the same assay performed on regular glass slides and static conditions. Moreover, limits of detection of 45 and 54 pg/ml were obtained for hepatitis B superficial antigen and HIV p24 antigen, respectively.