一种改进的双向电泳染色方法

本文涉及了双向电泳过程中的染色方法,即先用考马斯亮蓝染色,将胶上可见蛋白切下再银染的方法。这种方法可最大限度的减少胶中蛋白质点的损失,不仅避免了单一用考马斯亮蓝染色由于灵敏度不高而导致的低丰度蛋白的损失,也避免了单一用银染而使高丰度的蛋白因染色过度导致的损失。同时两种传统的染色方法结合完美,形成的新方法经济实用。     

A mechanically strong matrix for protein electrophoresis with enhanced silver staining properties.

Duracryl is a mechanically strong and elastic acrylamide-based matrix, useful for a wide variety of electrophoretic applications. The matrix is stable as a refrigerated solution for one year. Upon addition of appropriate catalysts, Duracryl forms a polymer-reinforced polyacrylamide gel matrix suitable for electrophoresis. The polymer-reinforced gel is superior to conventional polyacrylamide gels in terms of mechanical strength, elasticity and protein silver staining properties. Protein detection sensitivity by silver staining, as well as the linear response of silver deposition versus protein load, is equivalent to standard acrylamide/N,N'-methylene bisacrylamide gels. Additionally, the silver staining properties of the Duracryl matrix result in proteins appearing as monochromatic shades of grey instead of red, brown and yellow, as is the case of conventional polyacrylamide matrices. Monochromatic shades of grey are more suitable for image analysis and densitometry. The matrix is compatible with standard electroblotting and protein N-terminal sequencing procedures. Low acrylic acid content and conductivity allow incorporation of the matrix into isoelectric focusing gels. The matrix was found not to alter polypeptide migration relative to the standard acrylamide/N,N'-methylene bisacrylamide matrix.     

An improved method for the detection of genetic variations in DNA with denaturing gradient gel electrophoresis

We have examined the feasibility of denaturing gradient gel electrophoresis (DGGE) of RNA:DNA duplexes to detect variations in genomic and cloned DNAs. The result has demonstrated that employment of RNA:DNA duplexes makes DGGE much more practical for screening a large number of samples than that of DNA:DNA heteroduplexes originally developed by Lerman et al. (1986), because preparation of RNA probes is easier than that of DNA probes. Three different 32P-labeled RNA probes were produced. Genomic or cloned DNAs were digested with restriction enzymes and hybridized to labeled RNA probes, and resulting RNA:DNA duplexes were examined by DGGE. The presence of mismatch(es) was detected as a difference in mobility of bands on the gel. The experimental conditions were determined using DNA segments from cloned normal and 3 thalassemic human beta-globin genes. The results of the experiments on the cloned DNAs suggest that DGGE of RNA:DNA duplexes will detect nucleotide substitutions and deletions in DNA. In the course of these studies, a polymorphism due to a single-base substitution at position 666 of IVS2 (IVS2-666) of the human beta-globin gene was directly identified using genomic DNA samples. A study of 59 unrelated Japanese from Hiroshima was made in which the frequency of the allele with C at IVS2-666 was 0.48 and that of the allele with T was 0.52. This approach was found to be very effective for the detection of heritable variation and should be a powerful tool for the detection of fresh mutations in DNA, which occur outside the known restriction sites.     

Improved gel electrophoresis matrix for hydrophobic protein separation and identification

We propose an improved acrylamide gel for the separation of hydrophobic proteins. The separation strategy is based on the incorporation of N-alkylated and N,N′-dialkylated acrylamide monomers in the gel composition in order to increase hydrophobic interactions between the gel matrix and the membrane proteins. Focusing on the most efficient monomer, N,N′-dimethylacrylamide, the potentiality of the new matrix was evaluated on membrane proteins of the human colon HCT-116 cell line. Protein analysis was performed using an adapted analytical strategy based on FT-ICR tandem mass spectrometry. As a result of this comparative study, including advanced reproducibility experiments, more hydrophobic proteins were identified in the new gel (average GRAVY: −0.085) than in the classical gel (average GRAVY: −0.411). Highly hydrophobic peptides were identified reaching a GRAVY value up to 1.450, therefore indicating their probable locations in the membrane. Focusing on predicted transmembrane domains, it can be pointed out that 27 proteins were identified in the hydrophobic gel containing up to 11 transmembrane domains; in the classical gel, only 5 proteins containing 1 transmembrane domain were successfully identified. For example, multiple ionic channels and receptors were characterized in the hydrophobic gel such as the sodium/potassium channel and the glutamate or the transferrin receptors whereas they are traditionally detected using specific enrichment techniques such as immunoprecipitation. In total, membrane proteins identified in the classical gel are well documented in the literature, while most of the membrane proteins only identified on the hydrophobic gel have rarely or never been described using a proteomic-based approach.     

An improved horizontal slab gel electrophoresis apparatus for DNA separation

An improved horizontal slab gel electrophoresis apparatus was developed for the separation of DNA restriction fragments. The apparatus was designed for both analytical and preparative runs. The use of agarose or polyacrylamide wicks rather than paper wicks simplifies the use of and increases the capabilities of horizontal slab gel electrophoresis.     

DNA detection system using molecularly imprinted polymer as the gel matrix in electrophoresis

To develop a simple and inexpensive method for DNA detection, we prepared a molecularly imprinted polymer (MIP) for recognizing a specific double-stranded DNA (dsDNA) sequence and used it in an electrophoretic gel matrix. The MIP gel has many binding sites that are complementary in size, shape, and arrangement of functional groups of the target dsDNA sequence. During MIP gel electrophoresis (MIPGE), migration of the target dsDNA should be hindered by the capture effect of the binding sites in the MIP gel. This was confirmed by observation of deviations from the linear relationship between the migration distances of the DNA standard size markers in the polyacrylamide gel and those in the MIP gel. The migration distances of nontarget dsDNA maintained a linear relationship, however. In addition, the sequence selectivity of dsDNA in this method was investigated by using the Ha-ras gene and its point mutants. Except for A.T to T.A base pair substitution, mutant dsDNA (for example, substitution from A.T to C.G and from G.C to T.A) could be distinguished from the target (wild-type) dsDNA. Although some improvement in A.T (T.A) base pair distinction is still needed, this study is the first to demonstrate detection of a specific dsDNA sequence with MIPs and, as such, opens up a new realm for practical applications of MIPs.     

Direct detection of rutin-degrading isozymes with polyacrylamide gel electrophoresis

Rutin-degrading enzymes (RDEs) specifically hydrolyze the glycosidic linkages of rutin, producing quercetin and rutinose. Here we report a reliable and sensitive polyacrylamide gel electrophoresis and staining method for the detection of RDE isozymes, which is based on the aqueous solubility difference between rutin and quercetin, as well as the ultraviolet absorbance of quercetin. With this novel method, we achieved a detection limit of 12 ng with 107 U of RDE activity, enabling us to detect at least five RDE isozymes in tartary buckwheat seeds.     

Ultrasensitive staining-free protein detection after PAA gel electrophoresis using deep UV fluorescence

We present the observation of separated protein bands after polyacrylamide (PAA) gel electrophoresis based on the staining-free detection of their ultra violet (UV)-induced fluorescence employing deep UV confocal fluorescence microscopy. Mixtures of the three biological compounds beta-Galactosidase (from Escherichia coli), apo-Transferrin (bovine) and bovine serum albumin (BSA) have been separated and a staining free detection limit below 80 pg (7.0 x 10(8) molecules) per band has been achieved. This corresponds to approximately 270 molecules in the detection volume for confocal microscopy.     

Monitoring intracellular protein profiles with two-dimensional gel electrophoresis.

Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) is a method of separating complex protein mixtures, such as whole cell extracts, on the basis of protein isoelectric point and molecular weight. In bioprocess engineering, conventional 2D PAGE has tremendous potential to yield detailed information on the intracellular effect of various process conditions. It has been used in our work to examine global intracellular changes occurring in a typical cycloheximide fermentation and to look at the feedback regulatory behavior of cycloheximide biosynthesis. Application of the technique for bioprocess monitoring will require that the time necessary for preparation of a 2D electropherogram be substantially shortened. This may be accomplished by performing the separation on a miniature scale or eventually by use of capillary electrophoresis for one or more of the separations. Advantages and disadvantages of these two approaches are discussed.     

Three methods of capillary electrophoresis compared with high-resolution agarose gel electrophoresis for serum protein electrophoresis

We assessed the BioFocus 2000 capillary electrophoresis instrument for use in a routine clinical laboratory. We examined 210 serum samples received for serum protein electrophoresis by four methods: (1) The Bio-Rad HR015EC high-resolution serum protein kit on the BioFocus; (2) the Jenkins–Guerin (JG) method on the Applied Biosystems 270A HT Capillary Electrophoresis System (JG-ABI); (3) the Jenkins–Guerin method using the BioFocus (JG-BF); and (4) the quantitation of monoclonal bands found in 76 of the 210 samples was assayed by Helena Titan Hi-Res agarose gel electrophoresis (HRAGE). The correlation coefficient between the three sets of capillary electrophoresis monoclonal band results and the Helena quantitation was 0.92 or better. Although the quantitative comparison of monoclonal bands by HR015EC was very good, the lack of sharpness of monoclonal bands using the HR015EC kit meant our preference was to use the JG method on either the ABI or on the Biofocus.     

An improved method for separation of low-molecular-weight polypeptides by electrophoresis in sodium dodecyl sulfate-polyacrylamide gel

An improved system for SDS-polyacrylamide gel electrophoresis, capable of analyzing polypeptides having molecular weights from 1500 to 100,000 (especially showing high resolving power in the 1500 to 25,000 molecular weight range) is described. The 10 to 18% linear gradient gel containing 7 M urea with an acrylamide:bisacrylamide ratio of 20:1 and the Laemmli discontinuous buffer was used. The use of the gel with a high crosslinkage ratio is shown to be effective in lowering the leakage of low-molecular-weight polypeptides from the gel. This method has facilitated rapid detection of small amounts of low-molecular-weight polypeptides in body fluids by the use of silver stain. A procedure is presented for the elimination of false bands on the gel frequently encountered during silver staining. The separation patterns of enzymatic cleavage products of proteins, uremic plasma, and urines from nephropathy patients are illustrated. This system is also applicable in the separation of lipopolysaccharides and also for the detection of phospholipids.     

Gateway cloning is compatible with protein secretion from Pichia pastoris

Secretion of a recombinant protein from the yeast Pichia pastoris requires the presence of a signal peptide at the amino terminus. Maintaining the full amino acid sequence of the signal peptide is thought to be important for proper signal processing and protein secretion. We show that at least for one protein, a synthetic human interferon, the presence of a Gateway recombination site within the signal peptide is fully compatible with high levels of protein secretion. The amino termini of the secreted interferon proteins cloned with Gateway and cloned with restriction enzymes and ligase are identical, and the proteins were highly active in biological assays. Compatibility with Gateway cloning simplifies construction of plasmids directing secretion of recombinant proteins from P. pastoris.     

介绍一种改良的冷却装置的垂直板电泳槽

介绍一种改良的带冷却装置的浸入式垂直板电泳槽。用不会变形的有机玻璃制作固定架及控制凝胶厚度的隔条,以代替市售的可压缩的U形橡胶套,以螺丝固定玻璃板,用医用硅胶管密封以完全分隔正负极电泳缓冲液。经试用于蛋白质及核酸电泳,效果甚佳。     

Tissue proteomics by one-dimensional gel electrophoresis combined with label-free protein quantification

Label-free methods streamline quantitative proteomics of tissues by alleviating the need for metabolic labeling of proteins with stable isotopes. Here we detail and implement solutions to common problems in label-free data processing geared toward tissue proteomics by one-dimensional gel electrophoresis followed by liquid chromatography tandem mass spectrometry (geLC MS/MS). Our quantification pipeline showed high levels of performance in terms of duplicate reproducibility, linear dynamic range, and number of proteins identified and quantified. When applied to the liver of an adenomatous polyposis coli (APC) knockout mouse, we demonstrated an 8-fold increase in the number of statistically significant changing proteins compared to alternative approaches, including many more previously unidentified hydrophobic proteins. Better proteome coverage and quantification accuracy revealed molecular details of the perturbed energy metabolism.