Showing papers on "Gel electrophoresis published in 1994"
TL;DR: A two-dimensional native technique was developed that is suitable for preparation of highly pure membrane protein complexes and was applied to analysis of cytochrome bc/bf complexes.
1,223 citations
TL;DR: The unsurpassed qualitative and quantitative recovery of total RNA from cotton by hot borate buffer at alkaline pH, supplemented with PVP-40, deoxycholate, and/or NP-40 had also proven satisfactory for other recalcitrant plant species as well as for especially difficult tissue types.
924 citations
TL;DR: A simple, rapid, and selective procedure for extraction of the degraded, low-molecular-weight DNA from apoptotic cells, and the ladder pattern was detected from DNA extracted from 1-2 x 10(6) HL-60 cells, by flow cytometric criteria, as well as from blood and bone marrow samples from leukemic patients undergoing chemotherapy.
661 citations
TL;DR: The heme compound found in deoxyribonucleic acid (DNA) extracted from bloodstains, which is regarded as a major inhibitor of polymerase chain reaction (PCR), was characterized in comparison with alkaline and acid hematin, histidine and ammonia hemochromogens, and globin and serum albumin hemochROMogens digested by proteinase K.
Abstract: The heme compound found in deoxyribonucleic acid (DNA) extracted from bloodstains, which is regarded as a major inhibitor of polymerase chain reaction (PCR), was characterized in comparison with alkaline and acid hematin, histidine and ammonia hemochromogens, and globin and serum albumin hemochromogens digested by proteinase K. Alkaline and acid hematin were almost completely removed by phenol/chloroform treatment and ethanol precipitation, so as not to be copurified with DNA from the specimens. Spectrophotometric results indicated that the contaminant was likely to be the product of proteinase K digestion of some heme-blood protein complex, which was not completely extracted by organic solvents and remained in the ethanol precipitates of DNA. The results of polyacrylamide gradient gel electrophoresis and intensity of the inhibition of PCR suggested that the ligand of the contaminant was a somewhat large molecule, resistant to the proteolysis by proteinase K. The addition of bovine serum albumin to the reaction mixture prevented the inhibition of PCR by the heme compounds, probably by binding to the heme. This showed that the inhibition was not due to the irreversible inactivation of the enzyme.
534 citations
TL;DR: Transcriptional activity of CNLAC1 was found to be derepressed in the absence of glucose and to correspond to an increase in enzymatic activity.
Abstract: Melanin production is a major virulence factor for Cryptococcus neoformans, an organism causing life-threatening infections in an estimated 10% of AIDS patients. In order to characterize the events involved in melanin synthesis, an enzyme having diphenol oxidase activity was purified and its gene was cloned. The enzyme was purified as a glycosylated 75-kDa protein which migrated at 66 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after deglycosylation by endoglycosidase F. Substrate specificity resembled that of a laccase in that it oxidized multiple diphenolic and diamino compounds. Dopamine was shown by mass spectroscopy to be oxidized to decarboxy dopachrome, an intermediate of melanin synthesis. The enzyme contained 4.1 +/- 0.1 mol of copper per mol. It resembled a laccase in its absorbance spectrum, containing a peak of 610 nm and the shoulder at 320 nm, corresponding to the absorbance of a type I and type III copper, respectively. The cloned gene of C. neoformans laccase (CNLAC1) contained a single open reading frame encoding a polypeptide 624 amino acids in length. The encoded polypeptide contained a presumptive leader sequence, on the basis of its relative hydrophobicity and by comparison of the sequence to that of the N-terminal sequence of the purified enzyme. CNLAC1 also contained 14 introns ranging from 52 to 340 bases long. Transcriptional activity of CNLAC1 was found to be derepressed in the absence of glucose and to correspond to an increase in enzymatic activity.
355 citations
Journal Article•
338 citations
TL;DR: The electrophoretic method was used to analyze HA in two different liquid connective tissues and normal human knee joint synovial fluid showed a narrow HA molecular weight distribution, with a peak at 6-7 x 10(6).
293 citations
TL;DR: Experimental results indicated that the isolated hyperthermophilic archaeon strain, KOD1, was a thermostable thiol protease.
Abstract: A hyperthermophilic archaeon strain, KOD1, was isolated from a solfatara at a wharf on Kodakara Island, Kagoshima, Japan. The growth temperature of the strain ranged from 65 to 100 degrees C, and the optimal temperature was 95 degrees C. The anaerobic strain was an S0-dependent heterotroph. Cells were irregular cocci and were highly motile with several polar flagella. The membrane lipid was of the ether type, and the GC content of the DNA was estimated to be 38 mol%. The 16S rRNA sequence was 95% homologous to that of Pyrococcus abyssi. The optimum growth pH and NaCl concentration of the strain KOD1 were 7.0 and 3%, respectively. Therefore, strain KOD1 was identified as a Pyrococcus sp. Strain KOD1 produced at least three extracellular proteases. One of the most thermostable proteases was purified 21-fold, and the molecular size was determined to be 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 45 kDa by gel filtration chromatography. The specific activity of the purified protease was 2,160 U/mg of protein. The enzyme exhibited its maximum activity at approximately pH 7.0 and at a temperature of 110 degrees with azocasein as a substrate. The enzyme activity was completely retained after heat treatment at 90 degrees C for 2 h, and the half-life of enzymatic activity at 100 degrees C was 60 min. The proteolytic activity was significantly inhibited by p-chloromercuribenzoic acid or E-64 but not by EDTA or phenylmethylsulfonyl fluoride. Proteolytic activity was enhanced threefold in the presence of 8 mM cysteine. These experimental results indicated that the enzyme was a thermostable thiol protease.
287 citations
TL;DR: A novel form of an ATP-regulated, oligomeric, Ca(2+)-independent phospholipase A2 (iPLA2) has been purified from the cytosol of the murine macrophage-like cell line P388D1, indicating that the catalytically active iPLA2 exists as a large oligomersic complex, either through self-aggregation or association of the enzyme with other proteins.
260 citations
TL;DR: Reagents that improved specificity of the amplified product were formamide, glycerol, DMSO, Tween-20 and NP-40; on the other hand, urea, ethanol and 1-methyl-2-pyrrolidone (NMP) inhibited amplification.
237 citations
TL;DR: The results suggest that guanine quartets are not involved in the structure linking full-length, authentic genomic RNA of HIV-1 into a dimeric structure and suggest that a postassembly, PR-dependent maturation event is a common feature in genomic RNAs of retroviruses.
Abstract: We have characterized the dimeric genomic RNA in particles of both wild-type and protease (PR)-deficient human immunodeficiency virus type 1 (HIV-1). We found that the dimeric RNA isolated from PR- mutant virions has a lower mobility in nondenaturing gel electrophoresis than that from wild-type virions. It also dissociates into monomers at a lower temperature than the wild-type dimer. Thus, the dimer in PR- particles is in a conformation different from that in wild-type particles. These results are quite similar to recent findings on Moloney murine leukemia virus and suggest that a postassembly, PR-dependent maturation event is a common feature in genomic RNAs of retroviruses. We also measured the thermal stability of the wild-type and PR- dimeric RNAs under different ionic conditions. Both forms of the dimer were stabilized by increasing Na+ concentrations. However, the melting temperatures of the two forms were not significantly affected by the identity of the monovalent cation present in the incubation buffer. This observation is in contrast with recent reports on dimers formed in vitro from short segments of HIV-1 sequence: the latter dimers are specifically stabilized by K+ ions. K+ stabilization of dimers formed in vitro has been taken as evidence for the presence of guanine quartet structures. The results suggest that guanine quartets are not involved in the structure linking full-length, authentic genomic RNA of HIV-1 into a dimeric structure.
TL;DR: It is demonstrated that human stromelysin-3 acts as a powerful endopeptidase with a restricted substrate specificity distinct from all other matrix metalloproteinases, but also serve to identify serine proteinase inhibitors as potential physiologic targets at sites of extracellular matrix remodeling.
TL;DR: The alkaline microgel electrophoresis technique was modified to achieve a substantial increase in sensitivity, allowing the detection of DNA damage in the range of 50 mGy, and this increased sensitivity should extend the utility of this technique.
Abstract: The alkaline microgel electrophoresis technique was modified to achieve a substantial increase in sensitivity for the detection of radiation-induced DNA damage in human lymphocytes. This increased sensitivity was achieved through: (1) the addition of free radical scavengers to the electrophoresis solution to reduce DNA damage generated during alkaline unwinding and electrophoresis; (2) the modification of the electrophoresis unit to achieve a more uniform electric field; (3) the use of YOYO-1, a DNA dye, producing fluorescence 500-fold more intense than ethidium bromide; and (4) the introduction of an image analysis system for the quantitation of DNA migration. In addition to increasing sensitivity, these modifications have increased the speed with which observations can be quantified, and improved reproducibility from experiment to experiment. In human lymphocytes, these modifications have resulted in an increased sensitivity of several fold, allowing the detection of DNA damage in the range of 50 mGy. T...
TL;DR: It is found that the purified dystrophin-DAP complex was dissociated into several groups by n-octyl-beta-D-glucoside treatment, and it is concluded that A3b is a novel DAP different from 43DAG.
Abstract: Dystrophin is purified as a complex with several proteins from the digitonin-solubilized muscle cell membrane. Most of dystrophin-associated proteins (DAPs) are assumed to form a large oligomeric transmembranous glycoprotein complex on the sarcolemma and link dystrophin with a basement membrane protein, laminin. In the present study, we found that the purified dystrophin-DAP complex was dissociated into several groups by n-octyl-beta-D-glucoside treatment. In particular, we found that the glycoprotein complex stated above was dissociated into two distinct groups: one composed of 156DAG and 43DAG (A3a) and the other composed of 50DAG, 35DAG and A3b. We confirmed by crosslinking and immunoaffinity chromatography that these two groups existed in a complexes. We thus concluded that the glycoprotein complex consists of these two subcomplexes. Furthermore, A3b and 43DAG, which had been formerly treated simply as the 43DAG doublets due to their similar electrophoretic mobilities in SDS/PAGE, were shown to be present in two different subcomplexes. Based on the analyses by two-dimensional gel electrophoresis, peptide mapping and immunoblotting, we concluded that A3b is a novel DAP different from 43DAG.
TL;DR: In this article, a matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) of proteins, electroblotted onto polymer membranes after sodium dodecyl sulfate-polyacrylamide gel electropboresis separation is demonstrated.
Abstract: Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) of proteins, electroblotted onto polymer membranes after sodium dodecyl sulfate-polyacrylamide gel electropboresis (SDS-PAGE) separation is demonstrated. The proteins are desorbed directly from the blot membranes after matrix application. Desorption with 2.94-μm infrared radiation and succinic acid as matrix was found superior to 355-nm UV desorption using 2,5-dihydroxybenzoic acid as matrix. Several commercially available membranes tested resulted in protein signals after matrix incubation of the membrane. Systematic investigations for five different poly(vinylidene fluoride) (PVDF) membranes showed improved results for membranes exhibiting high specific surfaces
TL;DR: In this review DGGE and the several modifications of the original protocol are presented and its applications in human molecular genetics are summarized together with a preliminary comparison with other mutation detection technologies such as chemical cleavage, RNase protection, and single‐strand conformation polymorphism.
Abstract: The molecular analysis of genetic diseases relies on several technical approaches which allow genetic and physical mapping, characterization of the gene structure, expression studies, and identification of disease-causing mutations. Denaturing gradient gel electrophoresis (DGGE) allows the rapid screening for single base changes in enzymatically amplified DNA. The technique is based on the migration of double-stranded DNA molecules through polyacrylamide gels containing linearly increasing concentrations of a denaturing agent. In this review DGGE and the several modifications of the original protocol are presented. Moreover, its applications in human molecular genetics are summarized together with a preliminary comparison with other mutation detection technologies such as chemical cleavage, RNase protection, and single-strand conformation polymorphism.
TL;DR: It is concluded that cold-acclimated winter rye leaves produce multiple polypeptides with antifreeze activity that appear to be distinct from antifREEzes produced by fish and insects.
Abstract: Apoplastic extracts of cold-acclimated winter rye (Secale cereale L. cv Musketeer) leaves were previously shown to exhibit antifreeze activity. The objectives of the present study were to identify and characterize individual antifreeze proteins present in the apoplastic extracts. The highest protein concentrations and antifreeze activity were obtained when the leaf apoplast was extracted with ascorbic acid and either CaCl2 or MgSO4. Seven major polypeptides were purified from these extracts by one-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis under nonreducing conditions. The five larger polypeptides, of 19, 26, 32, 34, and 36 kD, exhibited significant levels of antifreeze activity, whereas the 11- and 13-kD polypeptides showed only weak activity. Five of these polypeptides migrated with higher apparent molecular masses on SDS gels after treatment with 0.1 M dithiothreitol, which indicated the presence of intramolecular disulfide bonds. The apparent reduction of the disulfide bonds did not eliminate antifreeze activity in four of the polypeptides that contained intramolecular disulfide bonds and exhibited significant levels of antifreeze activity. The amino acid compositions of these polypeptides were similar in that they were all relatively enriched in the residues Asp/Asn, Glu/Gln, Ser, Thr, Gly, and Ala; they all lacked His, except for the 26-kD polypeptide, and they contained up to 5% Cys residues. These polypeptides were examined with antisera to other cystine-containing antifreeze proteins from fish and insects, and no common epitopes were detected. We conclude that cold-acclimated winter rye leaves produce multiple polypeptides with antifreeze activity that appear to be distinct from antifreezes produced by fish and insects.
TL;DR: A transient entanglement coupling mechanism for DNA separation in dilute polymer solutions is developed, which suggests that there is no a priori upper size limit to DNA that can be separated by capillary electrophoresis in a constant field.
Abstract: Using capillary electrophoresis, large DNA molecules (2.0–23.1 kbp) may be rapidly separated in ultradilute polymer solutions (< 0.002% w/w) under, a high-voltage, steady field (265 V/cm). At this polymer concentration, the separation mechanism appears to be significantly different from that postulated to occur in crosslinked gels. Based on experimental results obtained with DNA restriction fragments and with negatively charged latex microspheres, we conclude that the Ogston and reptation models typically used to describe gel electrophoresis are not appropriate for DNA separations in such dilute polymer solutions. Electrophoresis experiments employing solutions of both small and large hydroxyethyl cellulose polymers highlight the importance of polymer length and concentration for the optimum resolution of DNA fragments varying in size from 72 bp to 23.1 kbp. A transient entanglement coupling mechanism for DNA separation in dilute polymer solutions is developed, which suggests that there is no a priori upper size limit to DNA that can be separated by capillary electrophoresis in a constant field.
TL;DR: A program is described which calculates the thermal stability and the denaturation behaviour of double-stranded DNAs and RNAs up to a length of 1000 base pairs based on recursive generation of conditional and a priori probabilities for base stacking.
Abstract: A program is described which calculates the thermal stability and the denaturation behaviour of double-stranded DNAs and RNAs up to a length of 1000 base pairs. The algorithm is based on recursive generation of conditional and a priori probabilities for base stacking. Output of the program may be compared directly to experimental results; thus the program may be used to optimize the nucleic acid fragments, the primers and the experimental conditions prior to experiments like polymerase chain reactions, temperature-gradient gel electrophoresis, denaturing-gradient gel electrophoresis and hybridizations. The program is available in three versions; the first version runs interactively on VAXstations producing graphics output directly, the second is implemented as part of the HUSAR package at GENIUSnet, the third runs on any computer producing text output which serves as input to available graphics programs.
TL;DR: A very simple device and method to prepare and manipulate horizontal polyacrylamide gels (H-PAGE), which eliminates almost all of the staff time taken in setup, loading, and recordkeeping and offers high resolution for genotyping pattern recognition.
TL;DR: A novel basic carboxypesptidase clearly different from carboxypeptid enzyme N has been isolated from human plasma and hydrolyzes the synthetic peptides hippuryl-L-arginine and hippuryL-lysine but, in contrast to other human basic car boxypePTidases, has only a limited esterase activity.
TL;DR: In apoptosis DNA degradation involves two distinct endonucleolytic activities, with only the first activity being essential for cell death.
TL;DR: Mouse sperm contain a major phosphotyrosine-containing protein which has been implicated as a sperm membrane receptor for the egg zona pellucida glycoprotein, ZP3 and this protein was purified and subjected to limited tryptic digestion and subsequent amino acid analysis, suggesting that the germ cell component of the testis possesses a unique tyrosine -phosphorylated form of hexokinase.
TL;DR: The breadth of the bands observed in separations of preformed DNA-dimeric dye complexes is due to the presence of DNA fragments with different numbers of bound dye molecules that can be resolved as closely spaced subbands in many of the separations.
Abstract: Fluorescence-detected capillary electrophoresis separations of [phi]X174/HaeIII DNA restriction fragments have been performed using monomeric and dimeric intercalating dyes. Replaceable hydroxyethyl cellulose solutions were used as the separation medium. Confocal fluorescence detection was performed following 488-nm laser excitation. The limits of DNA detection for on-column staining with monomeric dyes (ethidium bromide, two propidium dye derivatives, oxazole yellow, thiazole orange, and a polycationic thiazole orange derivative) were determined. The thiazole orange dyes provide the most sensitive detection with limiting sensitivities of 2-4 amol of DNA base pairs per band, and detection of the 603-bp fragment was successful, injecting from [phi]X174/HaeIII samples containing only 1-2 fg of this fragment per microliter. Separations of preformed DNA-dimeric dye complexes were also performed. The breadth of the bands observed in separations of preformed DNA-dimeric dye complexes is due to the presence of DNA fragments with different numbers of bound dye molecules that can be resolved as closely spaced subbands in many of our separations. The quality of these DNA-dye complex separations can be dramatically improved by performing the electrophoresis with 9-aminoacridine (9AA) in the column and running buffers. 43 refs., 10 figs., 1 tab.
TL;DR: Gel capillary-array electrophoresis has been developed to achieve a high-sensitivity and high-throughput DNA analysis using real-time fluorescence detection.
Abstract: Gel capillary-array electrophoresis has been developed to achieve a high-sensitivity and high-throughput DNA analysis using real-time fluorescence detection. To eliminate excitation light scattering at the capillary surfaces and to irradiate all the migration tracks simultaneously, the capillary tubes were removed from the irradiated region. DNA fragments were eluted from the gel capillaries and flowed into the lower open capillaries. Multiple sheath flows of buffer solution around all the capillaries were produced to prevent DNA band diffusion in the gel-free irradiated region. The fluorescence image was detected with a two-dimensional detector coupled with an image-splitting prism having four color filters
TL;DR: PCR-SSCP is a simple and rapid method for the detection and identification of a wide spectrum of bacteria by whole-cell-based PCR amplification with the use of conserved primers and identification by nondenaturing gel electrophoresis.
Abstract: A new molecular biological approach for the identification of bacteria is described. This approach employs PCR of bacterial cell lysates with conserved primers located in the 16S rRNA sequence flanking a variable region, and analysis of the amplified product was based on the principle of single-strand conformation polymorphism (SSCP). The PCR product was denatured and separated on a nondenaturing polyacrylamide gel. SSCP patterns were detected by silver staining the nucleic acids. The mobility of the single-stranded DNA is sequence dependent and could be used to identify the unknown bacteria. Feasibility of the technique was demonstrated for a broad panel of gram-negative and gram-positive bacteria. We tested over 100 strains of bacteria representing 15 genera and 40 species. With the use of only two primer sets, P11P-P13P and ER10-ER11, we were capable to discriminate the tested species at the genus and species levels. Species-specific patterns were obtained for, e.g., Clostridium spp., Listeria spp., Pseudomonas spp., and Enterobacter spp. PCR-SSCP is a sensitive technique; e.g., the sensitivity obtained for Escherichia coli cells was 30 CFU. This technique is a simple and rapid method for the detection and identification of a wide spectrum of bacteria by whole-cell-based PCR amplification with the use of conserved primers and identification by nondenaturing gel electrophoresis.
TL;DR: Albumin and α‐lipoprotein were negative acute phase proteins because their concentration significantly decreased during inflammation, and the concentration of α1‐acid glycoprotein, fetuin, β‐protease inhibitor, transferrin and α2‐macro‐globulins, as well as total proteins, did not change significantly during inflammation.
Abstract: Acute inflammation was induced in pigs using a single subcutaneous turpentine injection. The acute phase serum protein response was analyzed using crossed immunoelectrophoresis and immunodiffusion. The concentration of C reactive protein and haptoglobin increases 5–7 times 48 h after the injection, whereas the concentration of an α2-globulin, named pig major acute phase protein (pig-MAP), increases at least 15-fold. A molecular mass of 115 kDa for pig-MAP was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein did not crossreact with antisera to human hemopexin, ceruloplasmin, H-kininogen and complement factor C3. Albumin and α-lipoprotein were negative acute phase proteins because their concentration significantly decreased during inflammation. Finally, the concentration of α1-acid glycoprotein, fetuin, α1-protease inhibitor, transferrin and α2-macro-globulins, as well as total proteins, did not change significantly during inflammation.
TL;DR: The results described here extend the understanding of the structural potential of G-rich nucleic acids and may provide insight into the possible roles ofG-rich sequences and the novel structures they can form in biological systems.
Abstract: The telomeric DNA oligonucleotide 5'-G4T2G4-3' (Tet 1.5) spontaneously assembles into large superstructures we have termed G-wires. G-wires can be resolved by gel electrophoresis as a ladder pattern. The self-association of Tet 1.5 is noncovalent and exhibits characteristics of G4-DNA, a parallel four-stranded structure stabilized by guanine tetrads. Formation of G-wires is dependent upon the presence of Na+ and/or K+, and once formed, G-wires are resistant to denaturation. The results described here extend our understanding of the structural potential of G-rich nucleic acids and may provide insight into the possible roles of G-rich sequences and the novel structures they can form in biological systems.
TL;DR: A differential detergent fractionation (DDF) protocol is defined and characterized to enable protein dynamics in cytoskeletal and noncytOSkeletal compartments of isolated hepatocytes to be monitored simultaneously and reproducibly partitions hepatocytic proteins into functionally different compartments that are readily analyzed by 2‐D gel electrophoresis.
Abstract: Two-dimensional (2-D) gel electrophoresis is often used in toxicologic and metabolic studies to assess treatment- or stage-specific changes in protein synthesis, degradation or posttranslational modification. When combined with cell fractionation studies the detectability of low abundance proteins is enhanced, and changes in subcellular distribution of proteins can also be monitored. Detergent fractionation is a simpler alternative to differential pelleting, which partitions cellular constituents into functionally distinct populations while preserving cytoskeletal integrity. We defined and characterized a differential detergent fractionation (DDF) protocol to enable protein dynamics in cytoskeletal and noncytoskeletal compartments of isolated hepatocytes to be monitored simultaneously. Rat hepatocytes were maintained in suspension culture and fractionated by sequential extraction with detergentcontaining buffers (digitonin/EDTA, Triton/EDTA, Tween/deoxycholate). DDF reproducibly yielded four electrophoretically distinct fractions enriched in cytosolic, membrane-organelle, nuclear membrane and cytoskeletal-matrix markers, respectively. Immunoblotting with over 20 different antibodies corroborated the selectivity of fractionation and was used to characterize the distribution profiles of cytoskeletal (actin, tubulins, cytokeratins, vinculin, myosin, desmoplakins, fodrin, nuclear lamins) and noncytoskeletal proteins (heat-shock 70 proteins, glutathione-S-transferase, calpains, carbamoyl phosphate synthetase, etc.), as well as to identify spots in 2-D gels. Detergent buffers were compatible with equilibrium or nonequilibrium 2-D gel electrophoretic analysis. Extensive 2-D maps of acidic and basic proteins in each fraction were generated along with a tabular listing of Mr and pI. Thus, DDF reproducibly partitions hepatocytic proteins into functionally distinct cytoskeletal and noncytoskeletal compartments that are readily analyzed by 2-D gel electrophoresis. DDF is simple, applicable to use with other cell types or culture systems and is especially useful when biomaterial is limited (i.e., clinical studies).
TL;DR: A specific alanine racemase, which is a key enzyme in the biosynthesis of the undecapeptide cyclosporin A, was purified to electrophoretic homogeneity from the fungus Tolypocladium niveum, the first enzyme of this kind isolated from a eucaryotic organism.