Showing papers on "Gel electrophoresis published in 1996"

Mass Spectrometric Sequencing of Proteins from Silver-Stained Polyacrylamide Gels

Abstract: Proteins from silver-stained gels can be digested enzymatically and the resulting peptides analyzed and sequenced by mass spectrometry. Standard proteins yield the same peptide maps when extracted from Coomassie- and silver-stained gels, as judged by electrospray and MALDI mass spectrometry. The low nanogram range can be reached by the protocols described here, and the method is robust. A silver-stained one-dimensional gel of a fraction from yeast proteins was analyzed by nanoelectrospray tandem mass spectrometry. In the sequencing, more than 1000 amino acids were covered, resulting in no evidence of chemical modifications due to the silver staining procedure. Silver staining allows a substantial shortening of sample preparation time and may, therefore, be preferable over Coomassie staining. This work removes a major obstacle to the low-level sequence analysis of proteins separated on polyacrylamide gels.

Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry

Abstract: Molecular analysis of complex biological structures and processes increasingly requires sensitive methods for protein sequencing. Electrospray mass spectrometry has been applied to the high-sensitivity sequencing of short peptides, but technical difficulties have prevented similar success with gel-isolated proteins. Here we report a simple and robust technique for the sequencing of proteins isolated by polyacrylamide gel electrophoresis, using nano-electrospray tandem mass spectrometry. As little as 5 ng protein starting material on Coomassie- or silver-stained gels can be sequenced. Multiple-sequence stretches of up to 16 amino acids are obtained, which identify the protein unambiguously if already present in databases or provide information to clone the corresponding gene. We have applied this method to the sequencing and cloning of a protein which inhibits the proliferation of capillary endothelial cells in vitro and thus may have potential antiangiogenic effects on solid tumours.

Inhibition of NF-κB DNA Binding by Nitric Oxide

Abstract: It has been suggested that the NF-kappaB transcription factor family may mediate expression of the gene encoding the cytokine-inducible form of nitric oxide synthase (iNOS). To establish if nitric oxide (NO) could in turn affect activity of NF-kappaB, the ability of NO-donor compounds to influence NF-kappaB DNA binding activity in vitro was investigated. NO-donor compounds sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) both inhibited the DNA binding activity of recombinant NF-kappaB p50 and p65 homodimers and of p50-p65 heterodimers. Inhibition of NF-kappaB p50 DNA binding by NO-donor compounds involved modification of the conserved redox-sensitive C62 residue, as a C62S p50 mutant was significantly more resistant to SNP-mediated inactivation. Non-reducing SDS-polyacrylamide gel electrophoresis demonstrated that SNP could inhibit p50 DNA binding by mechanisms other than the formation of intersubunit disulphide bonds involving p50 residue C62. Electrospray ionization mass spectrometry of a synthetic NF-kappaB p5O peptide containing the C62 residue suggested that NO gas can modify C62 by S-nitrosylation. This study indicates that NO-donors can directly inhibit the DNA binding activity of NF-kappaB family proteins, suggesting that cellular NO provides another control mechanism for modulating the expression of NF-kappaB-responsive genes.

Phylogenetic compositions of bacterioplankton from two California estuaries compared by denaturing gradient gel electrophoresis of 16S rDNA fragments.

Abstract: The phylogenetic compositions of bacterioplankton assemblages from San Francisco Bay and Tomales Bay, Calif., differed substantially when analyzed by PCR-denaturing gradient gel electrophoresis; these differences are consistent with the results of previous studies demonstrating differences in their metabolic capabilities. PCR-denaturing gradient gel electrophoresis analysis of complex microbial assemblages was sensitive and reliable, and the results were reproducible as shown by experiments with constructed and naturally occurring assemblages.

Integrated Microdevice for DNA Restriction Fragment Analysis

Abstract: An integrated monolithic device (8 mm × 10 mm) that performs an automated biochemical procedure is demonstrated. The device mixes a DNA sample with a restriction enzyme in a 0.7-nL reaction chamber and after a digestion period injects the fragments onto a 67-mm-long capillary electrophoresis channel for sizing. Materials are precisely manipulated under computer control within the channel structure using electrokinetic transport. Digestion of the plasmid pBR322 by the enzyme HinfI and fragment analysis are completed in 5 min using 30 amol of DNA and 2.8 × 10-3 unit of enzyme per run.

Purification, characterization, molecular cloning, and expression of two laccase genes from the white rot basidiomycete Trametes villosa.

Abstract: Two laccases have been purified to apparent electrophoretic homogeneity from the extracellular medium of a 2,5-xylidine-induced culture of the white rot basidiomycete Trametes villosa (Polyporus pinsitus or Coriolus pinsitus). These proteins are dimeric, consisting of two subunits of 63 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and have typical blue laccase spectral properties. Under nondenaturing conditions, the two purified laccases have different pIs; purified laccase forms 1 and 3 have pIs of 3.5 and 6 to 6.5, respectively. A third purified laccase form 2 has the same N terminus as that of laccase form 3, but its pI is in the range of 5 to 6. The laccases have optimal activity at pH 5 to 5.5 and pH < or = 2.7 with syringaldazine and ABTS [2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid)] as substrates, respectively. The genes lcc1 and lcc2 coding for the two purified laccases (forms 1 and 3) have been cloned, and their nucleotide sequences have been determined. The genes for lcc1 and lcc2 have 8 and 10 introns, respectively. The predicted proteins are 79% identical at the amino acid level. From Northern (RNA) blots containing total RNA from both induced and uninduced cultures, expression of lcc1 is highly induced, while the expression of lcc2 appears to be constitutive. Lcc1 has been expressed in Aspergillus oryzae, and the purified recombinant protein has the same pI, spectral properties, stability, and pH profiles as the purified native protein.

Cold shock stress-induced proteins in Bacillus subtilis.

Abstract: Bacteria respond to a decrease in temperature with the induction of proteins that are classified as cold-induced proteins (CIPs). Using two-dimensional gel electrophoresis, we analyzed the cold shock response in Bacillus subtilis. After a shift from 37 to 15 degrees C the synthesis of a majority of proteins was repressed; in contrast, 37 proteins were synthesized at rates higher than preshift rates. One hour after cold shock, the induction of CIPs decreased, and after 2 h, general protein synthesis resumed. The identified main CIPs were excised from two-dimensional gels and were subjected to microsequencing. Three small acidic proteins that showed the highest relative induction after cold shock were highly homologous and belonged to a protein family of which one member, the major cold shock protein, CspB, has previously been characterized. Two-dimensional gel analyses of a cspB null mutant revealed that CspB affects the level of induction of several CIPs. Other identified CIPs function at various levels of cellular physiology, such as chemotaxis (CheY), sugar uptake (Hpr), translation (ribosomal proteins S6 and L7/L12), protein folding (PPiB), and general metabolism (CysK, Ilvc, Gap, and triosephosphate isomerase).

Capillary electrophoresis in analytical biotechnology

Abstract: Surface Modifications of Silica Walls: A Review of Different Methodologies, M. Chiari, M. Nesi, and P.G. Righetti Buffers, Electrolytes, and Additives for Capillary Electrophoresis, S. Moring Injection Methods in Capillary Electrophoresis, J.D. Olechno and J.A. Nolan Consecutive Protein Digestion and Peptide Derivatization Employing an On-Line Analyte Concentrator to Map Proteins Using Capillary Electrophoresis, N.A. Guzman Capillary Electrophoresis Interfaced with Mass Spectrometry: Electrospray Ionization and Continuous Flow Fast Atom Bombardment, K.B. Tomer, C.E. Parker, and L.J. Deterding Micellar Electrokinetic Chromatography in the Analysis of Amino Acids and Peptides, N. Matsubara and S. Terabe Analysis of rDNA Derived Proteins and Their Post-translational Modifications, K. Ganzler, N.W. Warne, and W.S. Hancock Capillary Zone Electrophoresis for the Analysis of Peptides, M. Castagnola, I. Messana, and D.V. Rossetti Analysis of Glycoproteins, Oligo- and Mono-saccharides, C. Chiesa, R.A. O'Neill, C. Horvath, and P.J. Oefner Capillary Electrophoresis of DNA, P.G. Righetti and C. Gelfi Principles of Size-based Separations in Polymer Solutions, J.-L. Viovy and C. Heller Isoelectric Focusing in Capillaries, P.G. Righetti, C. Gelfi, and M. Chiari Index

New insights into the composition, molecular mass and stoichiometry of the protein complexes of plant mitochondria

Abstract: Recently a powerful electrophoresis method for the native preparation and characterization of the respiratory protein complexes of mitochondria from fungi and mammals has been developed, which employs Coomassie dyes to introduce charge shifts on proteins (Schagger and von Jagow (1991) Anal Biochem 199, 223–231) The procedure, which is called ‘blue native-polyacrylamide gel electrophoresis’ (BN-PAGE), was modified and introduced for the analysis of mitochondria from higher plants BN-PAGE of mitochondrial protein from potato allows the separation of nine distinct protein complexes between 100 and 1000 kDa and reveals novel results for their composition, molecular mass and stoichiometry For the first time soluble mitochondrial protein complexes, like the HSP60 complex (750 kDa) and a complex of 200 kDa, which includes a formate dehydrogenase, are analysed by BN-PAGE Complex I from potato (1000 kDa) is about 100 kDa larger than the corresponding enzyme from beef and can be resolved into more than 30 different subunits on a second gel dimension The F1F0 ATP synthase (580 kDa) and the cytochrome c oxidase (160 kDa) from potato seem to contain more subunits than hitherto reported Direct sequencing of subunits revealed that the F1 part of the F1F0 ATP synthase lacks the oligomycin sensitivity conferring protein (OSCP), which was reported to be present in F1 parts of dicotyledonous plants, but contains the ATPase inhibitory protein N-terminal sequences of 16 mitochondrial proteins were obtained, several of which are presented for the first time from a plant source BN-PAGE allows the preparation of mitochondrial protein complexes from gram amounts of plant tissue, as the procedure only requires milligram amounts of organelles This potential of BN-PAGE is demonstrated by the separation and characterization of the mitochondrial enzyme complexes from Arabidopsis thaliana Further analysis of organellar protein complexes by BN-PAGE will allow the generation of ‘protein maps’ from different tissues and developmental stages or from mutant plants

Galactofuranose biosynthesis in Escherichia coli K-12: identification and cloning of UDP-galactopyranose mutase.

Abstract: We have cloned two open reading frames (orf6 and orf8) from the Escherichia coli K-12 rfb region. The genes were expressed in E. coli under control of the T7lac promoter, producing large quantities of recombinant protein, most of which accumulated in insoluble inclusion bodies. Sufficient soluble protein was obtained, however, for use in a radiometric assay designed to detect UDP-galactopyranose mutase activity (the conversion of UDP-galactopyranose to UDP-galactofuranose). The assay is based upon high-pressure liquid chromatography separation of sugar phosphates released from both forms of UDP-galactose by phosphodiesterase treatment. The crude orf6 gene product converted UDP-[alpha-D-U-14C]-galactopyranose to a product which upon phosphodiesterase treatment gave a compound with a retention time identical to that of synthetic alpha-galactofuranose-1-phosphate. No mutase activity was detected in extracts from cells lacking the orf6 expression plasmid or from orf8-expressing cells. The orf6 gene product was purified by anion-exchange chromatography and hydrophobic interaction chromatography. Both the crude extract and the purified protein converted 6 to 9% of the UDP-galactopyranose to the furanose form. The enzyme was also shown to catalyze the reverse reaction; in this case an approximately 86% furanose-to-pyranose conversion was observed. These observations strongly suggest that orf6 encodes UDP-galactopyranose mutase (EC 5.4.99.9), and we propose that the gene be designated glf accordingly. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified UDP-galactopyranose mutase revealed one major band, and analysis by electrospray mass spectrometry indicated a single major species with a molecular weight of 42,960 +/- 8, in accordance with that calculated for the Glf protein. N-terminal sequencing revealed that the first 15 amino acids of the recombinant protein corresponded to those expected from the published sequence. UV-visible spectra of purified recombinant enzyme indicated that the protein contains a flavin cofactor, which we have identified as flavin adenine dinucleotide.

Global analysis of proteins synthesized during phosphorus restriction in Escherichia coli.

Abstract: The pattern of proteins synthesized in Escherichia coli during steady-state growth in media with ample inorganic phosphate (Pi), upon limitation for Pi (without an alternative phosphorous compound), and during steady-state growth in media containing phosphonate (PHN) as the sole P source was examined by two-dimensional gel electrophoresis. Of 816 proteins monitored in these experiments, all those with differential synthesis rates greater than 2.0 or less than 0.5 upon phosphate limitation (P limitation) or during growth on PHN compared with their rates in the cultures with Pi were classified as belonging to the PL or PHN stimulon, respectively. The PL stimulon included 413 proteins, 208 showing induced synthesis and 205 showing repressed synthesis. The PHN stimulon was smaller: it included 257 proteins; 227 showed induced synthesis and 30 showed repressed synthesis. The overlap of the two stimulons included 137 proteins: most (118) were ones showing induced synthesis. The promoter regions of genes for several of the proteins with induced or repressed synthesis contained sequences which resembled the consensus sequence for PhoB binding. The aggregate mass of proteins responding to P limitation or growth on PHN was 30 to 40% of the cells' total mass. By comparing the proteins responding to P limitation with those responding to growth on PHN, one can speculate which proteins are likely involved in adapting cells to new P sources or in preparing cells to survive stationary phase.

Rapid preparation of bacterial DNA for pulsed-field gel electrophoresis.

Abstract: A disadvantage of genotyping bacterial strains by pulsed-field gel electrophoresis is that the procedure requires up to 6 days to complete. We modified a standard pulsed-field gel electrophoresis method (B.E. Murray, K.V. Singh, J.D. Health, B.R. Sharma, and G.M. Weinstock, J.Clin. Microbiol. 28:2059-2063, 1990) so that it could be completed in less than 3 days. We successfully applied this method to the analysis of a variety of gram-positive and gram-negative bacteria.

The major birch pollen allergen, Bet v 1, shows ribonuclease activity

Abstract: The major birch (Betula alba L.) pollen allergen, Bet v 1, has been shown to be homologous to pathogenesis-related proteins in a number of plants. Recently, it was demonstrated that a ginseng protein with high homology to an intracellular pathogenesis-related protein of parsley and to Bet v 1 is a ribonuclease (RNase). Birch pollen extract was separated in an RNase activity gel. Four major RNase bands were excised from the gel, reseparated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by Western blotting with a specific Bet v 1 monoclonal antibody and patient's serum. Thus the monomer and the dimer of Bet v 1 showed RNase activity. Purified recombinant Bet v 1 was shown to degrade plant RNA. The RNase activity of recombinant Bet v 1 was 180 units · mg−1.

Immunologic distribution of an organic anion transport protein in rat liver and kidney

Abstract: A Na(+)-independent organic anion transport protein was recently cloned from rat liver using a Xenopus laevis oocyte expression system [E. Jacquemin, B. Hagenbuch, B. Stieger, A.W. Wolkoff, and P.J. Meier, Proc. Natl. Acad. Sci. USA 91: 133-137, 1994]. Although expression of this protein is sufficient for cells to transport the organic anion bromosulfophthalein, little is known about its cell biology or biochemical characteristics. Northern blot analysis performed under high-stringency conditions revealed hybridization with RNA only from liver and kidney; transcripts appeared the same in these two organs. Within kidney, hybridization was greatest when RNA extracted from the outer medulla was used. Immunoblot analysis revealed that in liver, the transporter was enriched in 0.1 M Na2CO3-extracted membranes and sinusoidal plasma membrane preparations, consistent with its being an integral membrane protein. This 80-kDa protein migrated as a 65-kDa protein after treatment with N-glycanase. Immunomorphological examination of liver revealed basolateral plasma membrane localization. In 0.1 M Na2CO3-extracted membranes of kidney, the transporter migrated as an 83-kDa protein on nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). On reduction, it resolved into peptides of 33 and 37 kDa. SDS-PAGE migration of the liver protein was unaffected by reduction. Immunomorphological examination of kidney revealed apical plasma membrane localization in the S3 segment of the proximal tubule of the outer medulla. Differential processing and trafficking of this transporter in liver and kidney may have important functional and regulatory consequences.

Mobility Shift DNA‐Binding Assay Using Gel Electrophoresis

Abstract: The DNA-binding assay using nondenaturing polyacrylamide gel electrophoresis (PAGE) provides a simple, rapid, and extremely sensitive method for detecting sequence-specific DNA-binding proteins. Proteins that bind specifically to an end-labeled DNA fragment retard the mobility of the fragment during electrophoresis, resulting in discrete bands corresponding to the individual protein-DNA complexes. The assay described in this unit can be used to test binding of purified proteins or of uncharacterized factors found in crude extracts. This assay also permits quantitative determination of the affinity, abundance, association rate constants, dissociation rate constants, and binding specificity of DNA-binding proteins. Three additional protocols describe a competition assay using unlabeled competitor DNA, an antibody supershift assay, and multicomponent gel shift assays.

DNA sequencing by mass spectrometry

Abstract: Mass spectrometry has the potential to replace gel electrophoresis for fast DNA sequencing. In this tutorial paper, it is discussed how far mass spectrometry of DNA has come since the advent of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) and how much remains to be done before mass spectrometry will be a useful tool for DNA sequencing. A brief description of MALDI and ESI mass spectrometric analysis of DNA is presented along with the different strategies for DNA sequencing using mass spectrometry. These include mass spectrometric analysis to replace gel separation in Sanger sequencing, enzymatic ladder sequencing and sequencing by gas-phase fragmentation. The future role of mass spectrometry in DNA sequencing in the Human Genome Project and beyond is also discussed.

Expression of a Trichoderma reesei beta-xylanase gene (XYN2) in Saccharomyces cerevisiae.

Abstract: The XYN2 gene encoding the main Trichoderma reesei QM 6a endo-beta-1,4-xylanase was amplified by PCR from first-strand cDNA synthesized on mRNA isolated from the fungus The nucleotide sequence of the cDNA fragment was verified to contain a 699-bp open reading frame that encodes a 223-amino-acid propeptide The XYN2 gene, located on URA3-based multicopy shuttle vectors, was successfully expressed in the yeast Saccharomyces cerevisiae under the control of the alcohol dehydrogenase II (ADH2) and phosphoglycerate kinase (PGK1) gene promoters and terminators, respectively The 33-amino-acid leader peptide of the Xyn2 beta-xylanase was recognized and cleaved at the Kex2-like Lys-Arg residues, enabling the efficient secretion and glycosylation of the heterologous beta-xylanase The molecular mass of the recombinant beta-xylanase was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 27 kDa The construction of fur1 ura3 S cerevisiae strains allowed for the autoselection of the URA3-based XYN2 shuttle vectors in nonselective complex medium These autoselective S cerevisiae strains produced 1,200 and 160 nkat of beta-xylanase activity per ml under the control of the ADH2 and PGK1 promoters in rich medium, respectively The recombinant enzyme showed highest activity at pH 6 and 60 degrees C and retained more than 90% of its activity after 60 min at 50 degrees C

Molecular identification of bacteria from a coculture by denaturing gradient gel electrophoresis of 16S ribosomal DNA fragments as a tool for isolation in pure cultures.

Abstract: Molecular information about the bacterial composition of a coculture capable of sulfate reduction after exposure to oxic and microoxic conditions was used to identify and subsequently to isolate the components of the mixture in pure culture. PCR amplification of 16S ribosomal DNA fragments from the coculture, analyzed by denaturing gradient gel electrophoresis, resulted in two distinct 16S ribosomal DNA bands, indicating two different bacterial components. Sequencing showed that the bands were derived from a Desulfovibrio strain and an Arcobacter strain. Since the phylogenetic positions of bacteria are often consistent with their physiological properties and culture requirements, molecular identification of the two components of this coculture allowed the design of specific culture conditions to separate and isolate both strains in pure culture. This approach facilitates the combined molecular and physiological analysis of mixed cultures and microbial communities.