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1.
Genetic Variation in Proteins: Comparison of One-Dimensional and Two-Dimensional Gel Electrophoresis 总被引:2,自引:0,他引:2
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Two proteins with known characteristics on one-dimensional gels were studied by two-dimensional electrophoresis to compare the sensitivities of the two methods in detecting genetic variation. Two-dimensional electrophoresis was found to be less sensitive than several types of one-dimensional gels in distinguishing variants of both proteins. Denaturation of proteins in urea in the two-dimensional method makes it possible to distinguish closely related proteins that differ from each other by units of charge. Many more types of variation in protein sequences can be distinguished on one-dimensional gels in the absence of denaturants. The estimates of heterozygosity based on two-dimensional gels are lower than those based on other methods, at least in part, because of the limited types of sequence differences that can be detected on two-dimensional gels. The application of two-dimensional electrophoresis to the measurement of genetic variation and to the detection of new mutations should be made carefully, in view of the limited sensitivity of the method in finding differences in sequence. 相似文献
2.
Two-dimensional electrophoresis is an efficient method for the analysis of a broad range of complex protein samples. Current two-dimensional gel techniques are not suited for analysis of the small amount of proteins from tissue samples in the presence of high concentration of salts. Here we describe an improved two-dimensional gel electrophoresis procedure based on the use of a nonionic wetting agent, Tergitol NP7, in rehydration solution combined with the application of a linear potential sweep during isoelectrofocusing. This experimental approach yields a dramatic increase in the resolution and focusing of proteins visualized on two-dimensional gels. This technique is less time-consuming and laborious than the current techniques and can be used for a variety of two-dimensional electrophoresis applications, including proteome analysis. 相似文献
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小菜蛾血淋巴蛋白质双向电泳技术体系的建立及优化 总被引:1,自引:0,他引:1
采用不同方法对小菜蛾Plutella xylostella(L.)血淋巴进行双向电泳研究,建立了一套适用于小菜蛾血淋巴蛋白质组分析的双向电泳体系。从样品处理方案、等电聚焦时间、染色方法等因素对小菜蛾双向电泳结果的影响进行了分析和优化。结果表明,TCA/丙酮沉淀法提取蛋白损失少,图谱均匀清晰,分辨率和重复性更高。不同长度胶条的最佳上样量不同,胶条长度为7、17、24cm时,最佳上样量依次为20~50μg、50~300μg、100~500μg,而聚焦时间则分别以24000、50000、65000vhr为宜。第二向聚丙烯酰胺凝胶的浓度以12%为宜,电泳后蛋白点呈均匀分布。银染的灵敏度明显高于考马斯亮蓝染色,可以检测出更多的蛋白点,但考马斯亮蓝染色在后续蛋白点质谱鉴定中具有优势。 相似文献
5.
Protein Detection Methods in Proteomics Research 总被引:3,自引:0,他引:3
In proteomics research chemical as well as physical methods are used to detect proteins subsequently to their separation.
Physical methods are mostly applied after chromatography. They are either based on spectroscopy like light absorption at certain
wavelengths or mass determination of peptides and their fragments with mass spectrometry. Chemical methods are used after
two-dimensional electrophoresis and employ staining with organic dyes, metal chelates, fluorescent dyes, complexing with silver,
or pre-labeling with fluorophores. In some cases autoradiography is still used. Since all of these techniques are very different
in terms of sensitivity, their usefulness for quantitative determinations varies significantly. This review will describe
the various protein detection methods applied to electrophoresis gels. 相似文献
6.
Rae T Bonn R Lang E Stamenova S Burgos G Rupprecht K Fishpaugh J 《Analytical biochemistry》2011,(2):116-125
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. 相似文献
7.
A new type of two-dimensional electrophoresis for analysis of protein using cellulose acetate membrane has been developed. Prior to the separation, proteins in a sample are concentrated to a narrow zone on a strip of cellulose acetate according to “steady-state stacking” of isotachophoresis. Electroendosmotic counterflow on cellulose acetate membranes is advantageous for the isotachophoretic concentration of large sample volumes. The concentrated protein zone is then subjected to electrophoretic separation on the same strip. This first-dimensional separation including the concentrating process is named “concentrating electrophoresis.” Iso-electric focusing on several layers of cellulose acetate membrane is performed in the second-dimensional step. Many kinds of detection methods can be applied to the layers among which proteins are distributed. The novel two-dimensional electrophoresis takes only 5 h to perform. 相似文献
8.
Quantitative proteomics is one of the analytical approaches used to clarify crop responses to stress conditions. Recent remarkable advances in proteomics technologies allow for the identification of a wider range of proteins than was previously possible. Current proteomic methods fall into roughly two categories: gel-based quantification methods, including conventional two-dimensional gel electrophoresis and two-dimensional fluorescence difference gel electrophoresis, and MS-based quantification methods consists of label-based and label-free protein quantification approaches. Although MS-based quantification methods have become mainstream in recent years, gel-based quantification methods are still useful for proteomic analyses. Previous studies examining crop responses to stress conditions reveal that each method has both advantages and disadvantages in regard to protein quantification in comparative proteomic analyses. Furthermore, one proteomics approach cannot be fully substituted by another technique. In this review, we discuss and highlight the basis and applications of quantitative proteomic analysis approaches in crop seedlings in response to flooding and osmotic stress as two environmental stresses. 相似文献
9.
Ultra acidic proteins, generated by posttranslational modifications, are becoming increasingly important due to recent evidence showing their function as regulatory elements or as intermediates in degradation pathways in bacteria. Such proteins are important in neurodegenerative diseases and embryonic development, and they include the Alzheimer-related tau (τ) protein (resulting from posttranslational modifications) and the phosphor-storage embryonic proteins. The ultra acidic proteins are difficult to study because standard two-dimensional gel electrophoresis is inadequate for their analysis. Here we describe a novel electrophoresis system of anodic acidic gels that can replace isoelectric focusing as the first dimension of separation in two-dimensional electrophoresis. The system is based on a sodium acetate buffer (pH 4.6), is compatible with traditional stains (e.g., Coomassie blue) as well as novel fluorescent dyes (e.g., Pro-Q Diamond), and is quantitative for the analysis of ultra acidic proteins. The anodic acidic gels were used for the functional classification of the ultra acidic part of the Bacillus subtilis proteome, showing significant improvement over traditional two-dimensional electrophoresis. 相似文献
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Thorén K Gustafsson E Clevnert A Larsson T Bergström J Nilsson CL 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2002,782(1-2):219-226
Non-typable Haemophilus influenzae (NTHi) are small, gram-negative bacteria and are strictly human pathogens, causing acute otitis media, sinusitis and community-acquired pneumonia. There is no vaccine available for NTHi, as there is for H. influenzae type b. Recent advances in proteomic techniques are finding novel applications in the field of vaccinology. There are several protein separation techniques available today, each with inherent advantages and disadvantages. We employed a combined proteomics approach, including sequential extraction and analytical two-dimensional polyacrylamide electrophoresis (2D PAGE), and two-dimensional semi-preparative electrophoresis (2D PE), in order to study protein expression in the A4 NTHi strain. Although putative vaccine candidates were identified with both techniques, 11 of 15 proteins identified using the 2D PE approach were not identified by 2D PAGE, demonstrating the complementarily of the two methods. 相似文献
12.
How to Bring the “Unseen” Proteome to the Limelight via Electrophoretic Pre-Fractionation Techniques
The present review reports a panoply of electrophoretic methods as pre-fractionation tools in proteomic investigations in
preparation for mass spectrometry or two-dimensional electrophoresis map analysis. Such electrophoretic pre-fractionation
protocols include all those electrokinetic methodologies which are performed in free solution, most of them relying on isoelectric
focusing steps (although some approaches based on gels and granulated media are also discussed). Devices associated with electrophoretic
separations are multi-chamber apparatuses, such as the multi-compartment electrolyzers equipped with either isoelectric membranes
or with isoelectric beads, Off-Gel electrophoresis in a multi-cup device and the Rotofor, an instrument also based on a multi-chamber
system but exploiting the conventional technique of carrier-ampholyte-focusing. Other free-flow systems, as well as miniaturized
chambers, are also described. 相似文献
13.
Nowacka M Jackowiak P Rybarczyk A Magacz T Strozycki PM Barciszewski J Figlerowicz M 《Molecular biology reports》2012,39(1):139-146
The continuously growing interest in small regulatory RNA exploration is one of the important factors that have inspired the
recent development of new high throughput techniques such as DNA microarrays or next generation sequencing. Each of these
methods offers some significant advantages but at the same time each of them is expensive, laborious and challenging especially
in terms of data analysis. Therefore, there is still a need to develop new analytical methods enabling the fast, simple and
cost-effective examination of the complex RNA mixtures. Recently, increasing attention has been focused on the RNA degradome
as a potential source of riboregulators. Accordingly, we attempted to employ a two-dimensional gel electrophoresis as a quick
and uncomplicated method of profiling RNA degradome in plant or human cells. This technique has been successfully used in
proteome analysis. However, its application in nucleic acids studies has been very limited. Here we demonstrate that two dimensional
electrophoresis is a technique which allows one to quickly and cost-effectively identify and compare the profiles of 10–90
nucleotide long RNA accumulation in various cells and organs. 相似文献
14.
Preparative two-dimensional polyacrylamide gel electrophoresis of 32 P-labeled RNA 总被引:94,自引:0,他引:94
Complex mixtures of RNA molecules may be separated by two-dimensional electrophoresis on polyacrylamide gel slabs. The first dimension of the separation is carried out on acid gels in the presence of a high urea concentration, the second on more concentrated gels buffered at pH 8. The method has been applied to the complete separation of RNA fractions obtained after a preliminary gel electrophoresis of partial enzymic digests of 32P-labeled bacteriophage RNA. Another application is the fractionation of partial digests as obtained in sequence determination of RNA molecules. Spots are detected by autoradiography and extracted by a simple micro procedure which yields the material in a concentrated form suitable for sequence analysis by fingerprinting. 相似文献
15.
The intention of this review is to provide an overview of current methodologies employed in the rapidly developing field of ocular proteomics with emphasis on sample preparation, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS). Appropriate sample preparation for the diverse range of cells and tissues of the eye is essential to ensure reliable results. Current methods of protein staining for 2D-PAGE, protein labelling for two-dimensional difference gel electrophoresis, gel-based expression analysis and protein identification by MS are summarised. The uses of gel-free MS-based strategies (MuDPIT, iTRAQ, ICAT and SILAC) are also discussed. Proteomic technologies promise to shed new light onto ocular disease processes that could lead to the discovery of strong novel biomarkers and therapeutic targets useful in many ophthalmic conditions. 相似文献
16.
Nakul Mandal Steffen Heegaard Jan Ulrik Prause Bent Honoré Henrik Vorum 《Biological procedures online》2010,12(1):56-88
The intention of this review is to provide an overview of current methodologies employed in the rapidly developing field of
ocular proteomics with emphasis on sample preparation, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass
spectrometry (MS). Appropriate sample preparation for the diverse range of cells and tissues of the eye is essential to ensure
reliable results. Current methods of protein staining for 2D-PAGE, protein labelling for two-dimensional difference gel electrophoresis,
gel-based expression analysis and protein identification by MS are summarised. The uses of gel-free MS-based strategies (MuDPIT,
iTRAQ, ICAT and SILAC) are also discussed. Proteomic technologies promise to shed new light onto ocular disease processes
that could lead to the discovery of strong novel biomarkers and therapeutic targets useful in many ophthalmic conditions. 相似文献
17.
F. Wiederkehr 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1991,569(1-2)
The cerebrospinal fluid (CSF) is a specific ultrafiltrate of plasma, which surrounds the brain and spinal cord. The study of its proteins and their alteration may yield useful information on several neurological diseases. By using various electrophoretic separation techniques, several CSF proteins have been identified derived from plasma or from brain. Different one-dimensional methods, such as agarose gel electrophoresis and isoelectric focusing, are of similar value in identifying the non-specific oligoclonal bands, which are mainly helpful in the diagnosis of multiple sclerosis and other inflammatory diseases. Isoelectric focusing has a greater resolution than other one-dimensional methods, and it yields additional data about disease-associated proteins occurring in Alzheimer's disease, Huntington's chorea and amyotrophic lateral sclerosis. Silver-stained two-dimensional gels provide more information about the complex protein composition of CSF, particularly about proteins produced in the brain, such as apolipoprotein E and neuron-specific enolase. For the detection of oligoclonal antibodies, the investigation of protein changes revealed by Parkinson's disease, schizophrenia and Creutzfeldt—Jakob disease, and the analysis of CSF immune complexes, two-dimensional electrophoresis has a greater sensitivity. 相似文献
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Effect of various methods of preparation on the apparent protein composition of eukaryotic ribosomes. An essential preliminary to stoicheiometric measurements.
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1. We investigated whether there is any change in the relative amounts of ribosomal proteins during the isolation or extraction of the ribosomes by different methods, or during electrophoresis of the proteins. 2. To see whether proteins are lost (or gained) during the preparation of the ribosome we compared the two-dimensional protein pattern of three preparations: (a) ribosomes conventionally prepared by ultracentrifugation; (b) crude ribosomes obtained by pH5 precipitation; (c) crude ribosomes prepared by gel filtration. 3. To see whether proteins were lost during protein extraction we compared the two-dimensional pattern of ribosomes by using three different extraction methods (LiCl/urea, acetic acid and guanidine hydrochloride). 4. In all experiments listed above the relative amounts of the great majority of the proteins remained unchanged. We interpret this as showing that the relative amounts of ribosomal proteins (as we observed them on a two-dimensional gel) correspond to the proportions existing in the particle in vivo. 相似文献
20.
S B McGrath M Bounpheng L Torres M Calavetta C B Scott Y Suh D Rines N van Orsouw J Vijg 《Genomics》2001,78(1-2):83-90
Two-dimensional gene scanning (TDGS) is a method for analyzing multiple DNA fragments in parallel for all possible sequence variations, using extensive multiplex PCR and two-dimensional electrophoretic separation on the basis of size and melting temperature. High throughput application of TDGS is limited by the prolonged time periods necessary to complete the second-dimension electrophoretic separation step--denaturing gradient gel electrophoresis--and the current need for gel staining. To address these problems, we constructed a high-voltage, automatic, two-dimensional electrophoresis system and used this in combination with thinner gels to reduce two-dimensional electrophoresis time about 80%. Instead of gel staining, we used three different fluorophores to simultaneously analyze three samples in the same gel. These improvements greatly increase TDGS speed and throughput and make the method highly suitable for large-scale single-nucleotide polymorphism discovery and genetic testing. 相似文献